KR20240032971A - Optimized expression cassettes for gene therapy - Google Patents

Abstract

In some aspects, provided herein is a cardiac specific expression cassette. In some embodiments, provided herein is an expression cassette comprising a polynucleotide sequence encoding a gene product for the treatment of a cardiac disease, wherein the polynucleotide sequence comprises a promoter (e.g., a cardiac specific promoter), and an optional is operably linked to an enhancer (e.g., a cardiac specific enhancer). In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) virion, which encodes a capsid protein and a therapeutic gene product, e.g., a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter. A viral genome comprising an expression cassette comprising a polynucleotide sequence, wherein the expression cassette is flanked by inverted terminal repeats. The present disclosure further provides pharmaceutical compositions and methods for treating or preventing heart disease.

Description

Optimized expression cassettes for gene therapy

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 63/219,651, filed July 8, 2021, the entirety of which is incorporated herein by reference.

technology field

The present invention generally relates to gene therapy, e.g., optimized gene expression cassettes, recombinant adeno-associated virus (AAV) virions, and methods for using the same to treat and prevent heart disease.

Reference to Electronic Sequence Listing

The contents of the electronic sequence listing (TENA_021_02WO_SeqList_ST26.xml; size: 430,204 bytes; and creation date: July 8, 2022) are incorporated herein by reference in their entirety.

Cardiomyopathy accounts for approximately half of heart-related deaths. It is estimated that approximately 1 in 250 adults to 1 in 10,000 adults are affected by some form of cardiomyopathy (McKenna et al. Circ Res. 121:722-730 (2017)). Despite major efforts in screening, diagnosis, and treatment strategies, the prevalence of cardiomyopathy and the incidence of cardiomyopathy-related death remain high (Brieler et al. Am Fam physician . 96:640-646 (2017)).

Cardiomyopathy refers to a group of conditions of the heart that occur when its ability to pump blood is reduced. Decreased proper functioning of the heart muscle, such as contractile dysfunction, can lead to myocardial infarction, heart failure, blood clots, valve problems, and cardiac arrest. Cardiomyopathy can be separated into primary and secondary categories resulting in diverse phenotypes (McKenna et al . Circ Res. 121:722-730 (2017)). Primary cardiomyopathy may be genetic, acquired, or mixed in etiology. Genetic cardiomyopathies are inherited and include arrhythmogenic right ventricular dysplasia, hypertrophy, ion channel dysfunction, left ventricular densification, and mitochondrial myopathies. Acquired cardiomyopathies are primarily due to non-secondary, non-genetic causes that result in cardiac complications and include myocarditis, perinatal, tachycardia-induced cardiomyopathy, and stress-induced cardiomyopathy. Cardiomyopathy of mixed etiology is caused by a combination of non-genetic and genetic factors and includes dilated cardiomyopathy and restrictive cardiomyopathy. Secondary cardiomyopathy refers to heart disease due to non-cardiovascular causes. The underlying causes of secondary cardiomyopathy may be endocrine, infectious, exposure to toxins, autoimmune related, nutritional and/or neuromuscular.

Cardiomyocytes play a central role in cardiomyopathy. Cardiomyocytes, also called cardiac muscle cells, cardiomyocytes, or cardiomyocytes, are cardiac cells that make up the heart muscle and are responsible for the contractile function that allows the heart to function as a pump. There are many mechanisms that reduce the ability of cardiomyocytes to function properly (Dadson et al. Clin Sci (Lond) 131:1375-1392 (2017)). In arrhythmogenic right ventricular cardiomyopathy, the progressive transformation of cardiomyocytes into fibrotic tissue results in electrical isolation of cardiomyocytes and atrophy of the ventricular myocardium, the main structure responsible for the contractile function of the heart. In mitochondrial cardiomyopathy, deficiency in ATP production directly affects the contractile function of cardiomyocytes with high metabolic demands. Cardiomyopathy also occurs as a result of abnormal contractile function due to loss of normal Ca ²⁺ ion release, uptake, and sequestration processes due to loss of activity in regulatory enzymes such as sarco/endoplasmic reticulum calcium ATPase (SERCA) (Lennon et al. Int J Mol Med . 7:131-41 (2001)).

Treatment strategies for cardiomyopathy are needed.

Gene therapy approaches for the treatment of heart disease often use vectors constructed to transduce cardiac cells and express the transgene in a heart-tissue specific manner. Adeno-associated virus (AAV) vectors, cardiac-specific promoters, or a combination of both can be used to deliver polynucleotides encoding gene products (e.g., therapeutic proteins) to cardiac tissue, thereby treating cardiac disease. It can be used to express gene products.

However, achieving high expression of gene products remains a challenge, especially in cardiac cells.

Given these challenges, there remains a need in the art for improved gene therapy vectors, particularly for heart diseases.

In some aspects, the invention generally relates to a vector for delivering a polynucleotide encoding a dwarf open reading frame (DWORF) or other transgene to a cardiac cell, e.g., a cardiomyocyte. Disclosed herein are recombinant adeno-associated virus virions (rAAV virions) comprising an expression cassette and a capsid protein that effectively deliver DWORF polynucleotides into cardiac cells, along with associated compositions and methods. In any aspect described herein where reference is made to a DWORF transgene, DWORF may be replaced by a reference to another transgene whose expression is desired in cardiac cells. In some embodiments where reference is made to AAV based expression vectors and virions, the present disclosure also contemplates the use of other viral and non-viral vectors for delivery of transgenes. In particular, provided herein are certain viral and non-viral vectors that can be used to deliver transgenes into cardiac cells.

In some embodiments, provided herein is an expression cassette comprising a polynucleotide sequence, wherein the polynucleotide sequence:

i) one or more promoters, optionally one or more promoters being cardiac-specific promoters; and

ii) one or more copies of the transgene, optionally wherein the transgene encodes a polypeptide for treating or preventing heart disease or alleviating symptoms associated with heart disease;

wherein, in addition to elements (i) and (ii), the expression cassette is:

iii) one or more enhancers, optionally one or more enhancers being cardiac-specific enhancers;

iv) one or more copies of the transgene, wherein at least two copies of the transgene are present;

v) a polynucleotide sequence containing one or more introns; and/or

vi) comprises one or more of at least one copy of one or more copies of the transgene, which is codon optimized.

In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises 1, 2, 3 or 1 of elements (iii), (iv), (v), and (vi). Includes all four (any combination of elements (iii), (iv), (v) and (vi) may be used). In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises one or more enhancers, wherein the one or more enhancers are cardiac-specific enhancers and/or the polynucleotide sequence contains one or more introns. Includes. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises one or more enhancers, wherein the one or more enhancers are cardiac-specific enhancers and the polynucleotide sequence comprises one or more introns. do. In some embodiments, one or more introns can enhance or improve the efficiency of transgene expression. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of the transgene, wherein the two copies are not identical, and optionally the first copy is codon optimized. modified copy, and the second copy is not the codon optimized nucleotide sequence encoding the transgene. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of the transgene, wherein the two copies are not identical to each other, and optionally the first copy is codon is an optimized nucleotide sequence, the second copy is not a codon optimized nucleotide sequence encoding the transgene, and further the polynucleotide sequence includes one or more introns. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of the transgene, wherein the two copies are not identical to each other, and optionally the first copy is codon is an optimized nucleotide sequence, the second copy is not a codon-optimized nucleotide sequence encoding a transgene, and further the polynucleotide sequence comprises one or more introns, and further the polynucleotide sequence includes one or more enhancers (e.g., one or more enhancers are cardiac-specific enhancers). In some embodiments, one or more introns can enhance or improve the efficiency of transgene expression. In some embodiments in which two copies of the transgene are used, two copies of the promoter are also used.

In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more promoters, wherein one or more promoters are cardiac-specific enhancers. In some embodiments of the expression cassette, at least one promoter is a cardiac-specific promoter or all promoters are cardiac-specific promoters. In some embodiments of the expression cassette, the polynucleotide sequence includes a single promoter. In some embodiments of the expression cassette, the polynucleotide sequence includes two promoters. In some embodiments of the expression cassette, at least one of the one or more promoters is the chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments of the expression cassette, at least one of the one or more promoters is the human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the expression cassette includes a chicken cTnT promoter and a human cTnT promoter.

In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more copies of a transgene, wherein the transgene encodes a polypeptide to treat or prevent heart disease or alleviate symptoms associated with heart disease.

In some embodiments of the expression cassette, one or more copies of the transgene are at least two copies of the transgene. In some embodiments of the expression cassette, one or more copies of the transgene are two copies of the transgene. In some embodiments, one or more copies of the transgene are at least two copies of the transgene, and the polynucleotide sequence comprises at least two promoters each operably linked to at least two copies of the transgene. In some embodiments, one or more copies of the transgene are two copies of the transgene, and the polynucleotide sequence includes two promoters each operably linked to the two copies of the transgene. In some embodiments of an expression cassette comprising two copies of a transgene, the two “copies” are not identical. Without being bound by any theory, the use of two nucleic acid sequences encoding non-identical polypeptides can prevent DNA recombination within the vector. In some embodiments, the expression cassette includes one copy of the transgene with the original DNA sequence encoding the polypeptide and one copy of the transgene with the codon-optimized DNA sequence encoding the polypeptide. In some embodiments of an expression cassette comprising two copies of a transgene, the first copy of the transgene is sufficiently different from the second copy of the transgene to prevent DNA recombination.

In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more enhancers, and optionally, one or more enhancers are cardiac-specific enhancers. In some embodiments of the expression cassette, the polynucleotide sequence includes two or more enhancers (e.g., 2, 3, or 4 enhancers). In some embodiments of the expression cassette, one or more enhancers are cardiac-specific enhancers (e.g., at least one enhancer is a cardiac-specific enhancer, or 2, 3, or 4 or all enhancers are cardiac-specific enhancers) . In some embodiments of the expression cassette, the polynucleotide sequence includes one enhancer. In some embodiments of the expression cassette, the polynucleotide sequence does not include an enhancer. In some embodiments, the one or more cardiac specific enhancers are selected from ACTC1 enhancers and αMHC enhancers. In some embodiments, the ACTC1 enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78. In some embodiments, the ACTC1 enhancer comprises SEQ ID NO:78. In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. In some embodiments, the αMHC enhancer comprises SEQ ID NO:79. In some embodiments, the expression cassette includes an αMHC enhancer and an ACTC1 enhancer. In some embodiments of the expression cassette, the enhancer sequence comprises an αMHC enhancer followed by an ACTC1 enhancer. In some embodiments of the expression cassette, the enhancer sequence comprises an ACTC1 enhancer followed by an αMHC enhancer.

In some embodiments of the expression cassette, the polynucleotide sequence includes one or more introns. In some embodiments of the expression cassette, the polynucleotide sequence includes one intron. In some embodiments of the expression cassette, the polynucleotide sequence includes two introns. In some embodiments of the expression cassette, the polynucleotide sequence includes two or more introns. In some embodiments of the expression cassette, one or more introns are identical. In some embodiments of the expression cassette, one or more introns are different from each other. In some embodiments, the expression cassette includes an intron, and the intron is selected from a CMV intron and a chimeric intron. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80. In some embodiments, the CMV intron includes SEQ ID NO:80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. In some embodiments, the chimeric intron comprises SEQ ID NO:81. In some embodiments, the expression cassette includes a CMV intron and a chimeric intron. In some embodiments, the expression cassette does not contain an intron (e.g., does not contain a CMV intron or a chimeric intron).

In some embodiments of the expression cassette, at least one copy of one or more copies of the transgene is codon optimized (e.g., codon optimized for optimal human expression). In some embodiments of the expression cassette, two copies of the transgene are codon optimized. In some embodiments of the expression cassette, the first copy of the transgene is codon optimized and the second copy of the transgene is not codon optimized (e.g., is the original DNA sequence) or is otherwise different from the first copy. do. In some embodiments of the expression cassette, the first copy of the transgene is sufficiently different from the second copy of the transgene to prevent DNA recombination.

In some embodiments, the expression cassette further comprises one or more (e.g., two) post-transcriptional regulatory elements (“PTREs”). In some embodiments, the expression cassette further comprises one or more (e.g., two) WPRE sequences. In some embodiments, the expression cassette includes one WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26. In some embodiments, the WPRE sequence includes SEQ ID NO:26. In some embodiments, the expression cassette does not include a WPRE sequence.

In some embodiments, the expression cassette further comprises one or more polyadenylation sequences (“p(A)”). In some embodiments, the expression cassette includes one polyadenylation sequence. In some embodiments, the expression cassette includes two polyadenylation sequences. In some embodiments, the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28. In some embodiments, the expression cassette includes a BGH polyadenylation sequence and an SV40 polyadenylation sequence.

In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any of the following:

(i) 5'-promoter-intron-transgene-PTRE-p(A)-3';

(ii) 5'-promoter-transgene-PTRE-p(A)-promoter-transgene-PTRE-p(A);

(iii) 5'-Enhancer-Promoter-Transgene-PTRE-p(A)-3';

(iv) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-3';

(v) 5'-Enhancer-Enhancer-Promoter-Transgene-PTRE-p(A)-3';

(vi) 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-3';

(vii) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';

(viii) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';

(ix) 5'-p(A)-PTRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

(x) 5'-promoter-intron-transgene-PTRE-p(A)-p(A)-transgene-intron-promoter-3';

(xi) 5'-promoter-intron-transgene-PTRE-p(A)-promoter-intron-transgene-p(A)-3'; and

(xii) 5'-p(A)-PTRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

In some embodiments of the expression cassette, the transgene is attached to an expression cassette comprising a polynucleotide having an array of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. It has an increased expression level compared to In some embodiments, the increased expression level is about 1.5-fold to 150-fold. In some embodiments, the increased expression level is at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold.

In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

In some embodiments, the expression cassette comprises about 1.9 kb to about 3.7 kb. In some embodiments, the expression cassette comprises about 2.5 kb to about 3.7 kb, optionally about 2.8 kb to about 3.6 kb.

In some embodiments, the transgene within the expression cassette optionally encodes a polypeptide useful in the treatment of a cardiac disease or disorder when a wild-type copy of the gene is introduced into the subject. In some embodiments, the transgene within the expression cassette encodes a polypeptide associated with heart disease (e.g., when a loss-of-function mutation in the gene encoding the polypeptide is associated with heart disease).

In some embodiments, the transgene in the expression cassette is DWORF, JPH2, BAG3, CRYAB, lamin A isoform of LMNA, lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, DSP encodes a polypeptide selected from the DPII isoform, DSG2, and JUP. In some embodiments, the expression cassette is SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, or SEQ ID NO: 229 Contains transgenes that share . In some embodiments, the polypeptide is SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222 , at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, or SEQ ID NO: 230 Share.

In some embodiments, the transgene within the expression cassette encodes a DWORF polypeptide. In some embodiments, the transgene is at least 75%, 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 76, or SEQ ID NO: 77 %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the polypeptide is at least 75%, 80%, 90%, 95%, 96% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 43 %, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any of the following:

(i) 5'-human or chicken TnT promoter-chimeric intron-transgene-WPRE-p(A)-3';

(ii) 5'-first human or chicken TnT promoter-first copy of the transgene-WPRE-p(A)-second human or chicken TnT promoter-second copy of the transgene-WPRE-p(A) ( Optionally, wherein the first and second promoter sequences and the first and second copies of the transgene are in the same forward orientation;

(iii) 5'-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(iv) 5'-ACTC1e enhancer-heart-human TnT promoter-transgene-WPRE-bGHpA-3';

(v) 5'-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';

(vi) 5'-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(vii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(viii) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';

(ix) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';

(x) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(xi) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(xii) 5'-human TnT promoter-transgene with codon optimized polynucleotide sequence-WPRE-bGHpA-3';

(xiii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);

(xiv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation);

(xv) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence has) -SV40pA-3' (optionally, wherein the first transgene and the human TnT promoter are in reverse orientation and the second transgene and the chicken TnT promoter are in forward orientation);

(xvi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter- 3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation); and

(xvii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation); and (xix) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA. -3' (optionally, wherein the first transgene and the human TnT promoter are in reverse orientation and the second transgene and chicken TnT promoter are in forward orientation).

In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any of the following:

(i) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(ii) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';

(iii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);

(iv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation);

(v) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally, wherein the first transgene and the human TnT promoter are in reverse orientation and the second transgene and the chicken TnT promoter are in forward orientation);

(vi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter- 3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation); (vii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation); and

(viii) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene and human TnT promoter are in reverse orientation and the second transgene and chicken TnT promoter are in forward orientation).

In some embodiments, the expression cassette is a recombinant expression cassette.

In some embodiments, provided herein are recombinant vectors comprising any one of the expression cassettes described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

In some embodiments, provided herein is a recombinant adeno-associated virus (rAAV) virion comprising a capsid protein and a viral genome comprising any of the expression cassettes described herein, wherein the expression cassette includes an inverted terminal repeat (ITR). ) is located on the side. In some embodiments, the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the capsid protein is a chimeric capsid protein. In some embodiments, the capsid protein is an AAV5/AAV9 chimeric capsid protein. In some embodiments, the capsid protein is selected from any of SEQ ID NOs: 145-200.

In some embodiments, provided herein is a pharmaceutical composition comprising any one of the vectors described herein or any one of the rAAV virions described herein, and a pharmaceutically acceptable carrier.

In some embodiments, provided herein are kits comprising any of the pharmaceutical compositions described herein, or any component of such pharmaceutical compositions (e.g., vectors or rAAV virions).

In some embodiments, provided herein are methods of increasing expression of a polypeptide in cardiac cells or cardiac tissue, comprising using any of the vectors described herein, any rAAV virion described herein, or any of the rAAV virions described herein. It includes contacting the cell with the pharmaceutical composition. In some embodiments, the heart cells are cardiomyocytes. In some embodiments, the cardiac tissue is cardiac tissue. In some embodiments, polypeptide expression is increased by about 1.5-fold to 150-fold. In some embodiments, polypeptide expression is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

In some embodiments, provided herein are methods of increasing polypeptide expression in a subject, comprising using any of the vectors described herein, any rAAV virion described herein, or any pharmaceutical composition described herein. It includes administering to the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, following administration, polypeptide expression is increased in the heart of the subject. In some embodiments, the subject being treated has heart disease or is at risk for heart disease. In some embodiments, the subject being treated has borderline or reduced ejection fraction. In some embodiments, the subject being treated has a normal ejection fraction. In some embodiments, the subject being treated has a genetic mutation associated with heart disease (e.g., a mutation in the PLN gene). In some embodiments, the subject has low or undetectable levels of expression of the polypeptide encoded by the transgene compared to a healthy subject.

In some embodiments, provided herein are methods of treating or preventing a cardiac disease or disorder in a subject in need thereof, comprising using any of the vectors described herein, any of the rAAV virions described herein. , or administering to the subject any of the pharmaceutical compositions described herein. In some embodiments, the subject being treated has a heart disease or disorder. In some embodiments, the subject being treated is at risk of developing a heart disease or disorder. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the myocardial infarction is chronic myocardial infarction. In some embodiments, the subject has a genetic risk allele for a heart disease or disorder. In some embodiments, the subject has a genetic risk allele for a heart disease or disorder due to a genetic mutation. In some embodiments, the subject has a genetic risk allele for a heart disease or disorder due to a genetic mutation in the PLN gene (e.g., one or more mutations in the PLN gene described herein or known in the art) . In some embodiments, the cardiac disease or disorder is accompanied by reduced ejection fraction (HFrEF). In some embodiments, the cardiac disease or disorder is accompanied by preserved ejection fraction (HFpEF). In some embodiments, the method induces expression of a polypeptide encoded by the transgene in the heart of the subject. In some embodiments, the method induces expression of the polypeptide encoded by the transgene in cardiomyocytes of the subject. In some embodiments, the method does not result in detectable expression of the polypeptide encoded by the transgene in the subject's heart, liver, and/or muscles of the subject other than the subject's cardiac fibroblasts. In some embodiments, the method improves one or more measures of cardiac function, optionally fractional shortening and/or left ventricular internal dimension (LVID). In some embodiments, the improvement in cardiac function is at week 2, week 4, week 6, week 8, week 10, week 12, week 14, week 16, week 18, week 20, week 22, and/or week 24 after administration. or observed later. In some embodiments, administration is systemic administration. In some embodiments, systemic administration is selected from intravenous or intraluminal injection. In some embodiments, when rAAV virions are administered, they are administered as a unit dose. In some embodiments, the unit dosage is about 3Х10 ¹⁴ vg/kg or less, about 2Х10 ¹⁴ vg/kg or less, about 1Х10 ¹⁴ vg/kg or less, about 9Х10 ¹³ vg/kg or less, about 8Х10 ¹³ vg/kg or less, about 7Х10 ¹³ vg/kg or less, about 6Х10 ¹³ vg/kg or less, about 5Х10 ¹³ vg/kg or less, about 4Х10 ¹³ vg/kg or less, about 3Х10 ¹³ vg/kg or less, about 2Х10 ¹³ vg/kg or less, or about 1Х10 Contains less than ¹³ vg/kg. In some embodiments, the subject being treated is a mammal. In some embodiments, the subject being treated is a human.

In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid protein and an expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter. A viral genome comprising, wherein the expression cassette is flanked by inverted terminal repeats.

In some embodiments, the DWORF polypeptide is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43. Share. In some embodiments, the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

In some embodiments, the promoter is the chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments, the promoter is the human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

In some embodiments, the expression cassette further comprises one or more enhancers. In some embodiments, the one or more enhancers are selected from the ACTC1 cardiac enhancer and the αMHC enhancer. In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO:78. In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. In some embodiments, the αMHC enhancer comprises SEQ ID NO:79.

In some embodiments, the expression cassette further includes an intron. In some embodiments, the intron is selected from CMV introns and chimeric introns. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80. In some embodiments, the CMV intron includes SEQ ID NO:80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. In some embodiments, the chimeric intron comprises SEQ ID NO:81.

In some embodiments, the expression cassette further comprises a WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26. In some embodiments, the WPRE sequence includes SEQ ID NO:26.

In some embodiments, the expression cassette further comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28.

In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

In some embodiments, the expression cassette includes a single promoter. In some embodiments, the expression cassette includes two promoters. In some embodiments, the expression cassette comprises a single copy sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette includes two copies of the sequence encoding the DWORF polypeptide. In some embodiments, when an expression cassette comprises two copies of a sequence encoding a DWORF polypeptide, the two “copies” are not identical. Without being bound by any theory, the use of two nucleic acid sequences encoding non-identical polypeptides can prevent DNA recombination within the vector. In some embodiments, the expression cassette includes one copy with the original DNA sequence encoding the DWORF polypeptide and one copy with the codon-optimized DNA sequence encoding the DWORF polypeptide. In some embodiments, the expression cassette comprises two copies of the sequence encoding the DWORF polypeptide, with one copy being codon optimized and one copy not being codon optimized. In some embodiments, the expression cassette includes 1, 2, 3, or 4 enhancers. In some embodiments, the expression cassette includes 1 or 2 introns. In some embodiments, the expression cassette includes 1 or 2 WPRE sequences. In some embodiments, the expression cassette includes 1 or 2 polyadenylation sequences.

In some embodiments, the expression cassette comprises no more than about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb. In some embodiments, the expression cassette is about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb. kb, about 3.1 kb, about 3.2 kb, or more.

In some embodiments, the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. Contains polynucleotide sequences that share % identity. In some embodiments, the expression cassette comprises either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61. In some embodiments, the expression cassette includes SEQ ID NO:61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:62. In some embodiments, the expression cassette includes SEQ ID NO:62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:63. In some embodiments, the expression cassette includes SEQ ID NO:63.

In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the capsid protein is a chimeric capsid protein. In some embodiments, the capsid protein is an AAV5/AAV9 chimeric capsid protein. In some embodiments, the capsid protein is selected from any of SEQ ID NOs: 145-200.

In one aspect, the present disclosure provides an expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter. In some embodiments, the DWORF polypeptide is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43. share the same identity. In some embodiments, the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

In some embodiments, the promoter is the chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments, the promoter is the human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

In some embodiments, the expression cassette further comprises one or more enhancers. In some embodiments, the one or more enhancers are selected from the ACTC1 cardiac enhancer and the αMHC enhancer. In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO:78. In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. In some embodiments, the αMHC enhancer comprises SEQ ID NO:79.

In some embodiments, the expression cassette further includes an intron. In some embodiments, the intron is selected from CMV introns and chimeric introns. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80. In some embodiments, the CMV intron includes SEQ ID NO:80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. In some embodiments, the chimeric intron comprises SEQ ID NO:81.

In some embodiments, the expression cassette further comprises a WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26. In some embodiments, the WPRE sequence includes SEQ ID NO:26.

In some embodiments, the expression cassette further comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28.

In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

In some embodiments, the expression cassette includes a single promoter. In some embodiments, the expression cassette includes two promoters. In some embodiments, the expression cassette comprises a single copy sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette includes two copies of the sequence encoding the DWORF polypeptide. In some embodiments, the expression cassette includes 1, 2, 3, or 4 enhancers. In some embodiments, the expression cassette includes 1 or 2 introns. In some embodiments, the expression cassette includes 1 or 2 WPRE sequences. In some embodiments, the expression cassette includes 1 or 2 polyadenylation sequences.

In some embodiments, the expression cassette comprises no more than about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb. In some embodiments, the expression cassette is about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb. kb, about 3.1 kb, about 3.2 kb, or more.

In some embodiments, the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. Contains polynucleotide sequences that share % identity. In some embodiments, the expression cassette comprises either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61. In some embodiments, the expression cassette includes SEQ ID NO:61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:62. In some embodiments, the expression cassette includes SEQ ID NO:62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:63. In some embodiments, the expression cassette includes SEQ ID NO:63.

In some embodiments, the expression cassette comprises a 5' inverted terminal repeat and a 3' inverted terminal repeat.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a rAAV virion disclosed herein and a pharmaceutically acceptable diluent. In another aspect, the present disclosure provides kits comprising the pharmaceutical compositions provided herein.

In one aspect, the disclosure provides a method of increasing DWORF expression in a cell, the method comprising contacting the cell with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cells are cardiac cells. In some embodiments, the heart cells are cardiomyocytes. In some embodiments, DWORF expression is increased about 1.5-fold to 150-fold. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

In one aspect, the disclosure provides a method of increasing DWORF expression in a tissue, the method comprising contacting the tissue with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, DWORF expression is increased about 1.5-fold to 150-fold. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo.

In one aspect, the disclosure provides a method of increasing DWORF expression in an organ, the method comprising contacting the organ with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, DWORF expression is increased about 1.5-fold to 150-fold.

In some embodiments, the organ is the heart. In some embodiments, the heart has heart disease or is at risk for heart disease. In some embodiments, the heart has a reduced or borderline ejection fraction. In some embodiments, the heart has a normal ejection fraction.

In some embodiments, the heart contains a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable DWORF expression compared to a healthy heart. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

In one aspect, the disclosure provides a method of increasing DWORF expression in a subject, the method comprising administering to the subject a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, DWORF expression is increased in the heart of the subject. In some embodiments, the subject has heart disease or is at risk for heart disease. In some embodiments, the subject has borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to a healthy subject.

In one aspect, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising administering to the subject a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. do.

In some embodiments, the subject has a heart disease or disorder. In some embodiments, the subject is at risk of developing a heart disease or disorder. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the heart disease or disorder is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the heart disease or disorder is chronic myocardial infarction. In some embodiments, the heart disease or disorder is acute myocardial infarction.

In some embodiments, the subject has a genetic risk allele for a heart disease or disorder. In some embodiments, the genetic risk allele comprises a mutation to the PLN gene. In some embodiments, the mutation to the PLN gene is a PLN promoter mutation. In some embodiments, the mutation to the PLN gene is a PLNL39stop mutation. In some embodiments, the mutation to the PLN gene is an RC9 mutation. In some embodiments, the mutation to the PLN gene is an R9L mutation. In some embodiments, the mutation to the PLN gene is a PLN gene duplication. In some embodiments, the mutation in the PLN gene is an R14del mutation.

In some embodiments, the cardiac disease or disorder is accompanied by reduced ejection fraction (HFrEF). In some embodiments, the cardiac disease or disorder is accompanied by preserved ejection fraction (HFpEF).

In some embodiments, the method causes expression of a DWORF polypeptide in the heart of the subject. In some embodiments, the method results in expression of a DWORF polypeptide in cardiomyocytes.

In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in muscles of the subject other than the heart. In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in the liver of the subject. In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in cardiac fibroblasts.

In some embodiments, the method improves one or more measures of cardiac function, optionally fractional shortening and/or left ventricular internal dimension (LVID). In some embodiments, improvement in cardiac function is observed from week 2 to week 16. In some embodiments, the method reduces cardiac remodeling. In some embodiments, the method responds to a decrease in DWORF expression in a subject suffering from or at risk of heart disease.

In some embodiments, rAAV virions are administered by systemic administration. In some embodiments, systemic administration is selected from intravenous or intraluminal injection.

In some embodiments, rAAV is administered as a unit dose. In some embodiments, the unit dosage is about 3Х10 ¹⁴ vg/kg or less, about 2Х10 ¹⁴ vg/kg or less, about 1Х10 ¹⁴ vg/kg or less, about 9Х10 ¹³ vg/kg or less, about 8Х10 ¹³ vg/kg or less, about 7Х10 ¹³ vg/kg or less, about 6Х10 ¹³ vg/kg or less, about 5Х10 ¹³ vg/kg or less, about 4Х10 ¹³ vg/kg or less, about 3Х10 ¹³ vg/kg or less, about 2Х10 ¹³ vg/kg or less, or about 1Х10 Contains less than ¹³ vg/kg.

In one aspect, the disclosure provides a method of alleviating one or more symptoms of a cardiac disease or disorder in a subject in need thereof, the method comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. Includes.

In one aspect, the present disclosure provides a method of improving one or more symptoms of a cardiac disease or disorder in a subject in need thereof, the method comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. Includes.

In one aspect, the disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject in need thereof, the method comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. Includes.

In one aspect, the present disclosure provides an expression cassette comprising a polynucleotide comprising an array of elements from 5' to 3', wherein the elements include: i) one or more promoters; ii) optionally one or more enhancers; iii) optionally one or more introns; iv) one or more transgenes; v) optionally one or more WPRE sequences; and vi) optionally one or more polyadenylation sequences, p(A). In some embodiments, the arrangement of the elements from 5' to 3' is selected from: i) 5'-promoter-intron-transgene-WPRE-p(A)-3'; ii) 5'-Enhancer-Promoter-Transgene-WPRE-p(A)-3'; iii) 5'-Enhancer-Enhancer-Promoter-Transgene-WPRE-p(A)-3'; iv) 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3'; v) 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3'; vi) 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3'; vii) 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p (A)-Enhancer-Promoter-Intron-Transgene-p(A)-3'; viii) 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3'; ix) 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3'; x) 5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; and xi) 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'. In some embodiments, the transgene is compared to a second expression cassette comprising a polynucleotide having an array of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. has increased expression levels. In some embodiments, the increased expression level is from about 1.5-fold to about 150-fold compared to the second expression cassette.

In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid protein and a viral genome comprising an expression cassette of any of those disclosed herein, the expression cassette being flanked by inverted Terminal repeats are located. In some embodiments, the expression cassette includes a transgene, wherein the transgene encodes a polypeptide for use in treating or preventing heart disease or alleviating symptoms associated with heart disease. In some embodiments, the capsid protein is selected from any of SEQ ID NOs: 145-200.

Figure 1 depicts a schematic diagram of an exemplary embodiment of an expression cassette comprising a polynucleotide encoding a promoter, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.
Figure 2 is a graph showing the expression of GFP delivered to human induced pluripotent stem cell derived cardiomyocytes in vitro using an embodiment of an expression cassette packaged within a rAAV virion.
Figure 3 is a graph showing expression of human DWORF polypeptides delivered in vivo in a murine model using one embodiment of an expression cassette packed into rAAV virions using AAV9 capsid protein.
Figure 4A is a graph showing expression of human DWORF in induced pluripotent stem cell derived cardiomyocytes using one embodiment of an expression cassette packaged within a rAAV virion, one of several AAV protein capsid proteins described herein. .
Figure 4B is a series of images showing the expression of GFP using one embodiment of the expression cassette described herein packaged within a rAAV virion, one of several AAV protein capsid proteins described herein.
Figure 5A is a graph showing DWORF RNA expression in heart tissue of animals treated with rAAV virions containing an embodiment of an expression cassette packaged with one of the five chimeric capsid proteins or the AAV9 capsid protein.
Figure 5B is a graph showing DWORF protein levels in heart tissue of animals treated with rAAV virions containing an embodiment of an expression cassette packaged with one of the four chimeric capsid proteins or the AAV9 capsid protein.
Figure 6A is a graph showing improved ejection fraction in the PLN-R14 ^β / ^β mouse model following treatment with one embodiment of an expression cassette packaged within rAAV virions using AAV9 protein capsid.
Figure 6B is a graph showing fractional shortening in the PLN-R14 ^β / ^β mouse model following treatment with one embodiment of an expression cassette packaged within a rAAV virion using an AAV9 protein capsid.
Figure 7A depicts a schematic diagram of an exemplary expression cassette orientation comprising a polynucleotide encoding a promoter, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.
Figure 7B depicts a schematic diagram of an exemplary expression cassette orientation comprising a polynucleotide encoding a promoter, one or more enhancers, an intron, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats. .
Figure 7C depicts a schematic diagram of an exemplary expression cassette orientation comprising a polynucleotide encoding a promoter, one or more enhancers, an intron, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats. .
Figure 8A is a schematic diagram outlining the strategy for assessing the expression of DWORF in cardiomyocyte mice retro-orbitally injected with AAV9:DWORF constructs containing various regulatory elements and sequences in vivo.
Figure 8B is a Western blot showing the expression of DWORF and GAPDH in cardiomyocyte mice retro-orbitally injected with AAV9:DWORF constructs containing various regulatory elements and sequences in vivo.
Figure 8C is a chart showing DWORF expression levels in animal models achieved using a panel of rAAV virions containing expression cassettes encoding DWORF polypeptides.
Figure 9 is a plot showing improved ejection fraction in an animal model of cardiomyopathy treated with a panel of rAAV virions containing an expression cassette encoding a DWORF polypeptide.
Figure 10 is a plot showing preserved ejection fraction in an animal model of cardiomyopathy treated with a panel of rAAV virions containing an expression cassette encoding a DWORF polypeptide.
Figure 11A is a schematic diagram of DWORF gene therapy efficacy study in the MLP-KO DCM mouse model.
Figures 11B and 11C demonstrate that the AAV9:DWORF construct containing the novel promoter improves ejection fraction compared to saline controls in the MLP-KO DCM mouse model.
Figures 11D and 11E demonstrate that the AAV9:DWORF construct improved exercise capacity, including running distance and time to exhaustion, in the MLP-KO DCM mouse model at 26 weeks post-treatment.
Figure 12A is a schematic detailing the DWORF gene therapy tolerability study in untreated mice.
Figure 12B demonstrates that AAV9:pHZ21 is well tolerated in untreated mice up to doses of 2 x 10 ¹⁴ vg/kg with no differences in body weight, ejection fraction, heart rate, and left ventricular mass (LV mass).

In some embodiments, optimized gene therapy expression cassettes, and their use in the treatment of heart disease are described herein. In some embodiments, gene therapy expression cassettes capable of mediating high expression of a transgene are described herein. In some embodiments, much higher transgene expression than can be achieved using the cTnT promoter alone (e.g., the chicken cTnT promoter alone and/or the human cTnT promoter alone) or using the expression cassette shown in Figure 7A. Described herein are cardiac specific gene therapy expression cassettes that can mediate. In some embodiments, gene therapy expression cassettes are described herein that can lower viral load while achieving desired expression of a transgene. In some embodiments, gene therapy expression cassettes are described herein that allow to achieve sustained expression of a transgene. In some embodiments, after administering a gene therapy expression cassette comprising a transgene to a subject, expression of the transgene occurs for at least, or at least 12, 16, 18, 20, 22, 24, or 26 weeks. Gene therapy expression cassettes that enable expression to be achieved are described herein. In some embodiments, a gene therapy expression cassette is disclosed herein that allows to achieve expression of the transgene for at least 24 weeks, or at least 6 months, after administering the gene therapy expression cassette comprising the transgene to a subject. It is described. In some embodiments, administration to a subject of a gene therapy expression cassette described herein improves one or more cardiac functions (e.g., improves ejection fraction or improves exercise capacity). In some embodiments, administration to a subject of a gene therapy expression cassette described herein results in a sustained improvement in cardiac function (e.g., a sustained improvement in ejection fraction or a sustained improvement in exercise capacity). In some embodiments, administration to a subject of a gene therapy expression cassette described herein involves one or more cardiac Improve functionality. In some embodiments, administration to a subject of a gene therapy expression cassette described herein improves one or more cardiac functions for at least, or at least 24 weeks, or at least 6 months after administration. In some embodiments, the gene therapy expression cassettes described herein allow for cardiac cell-specific expression of a transgene (e.g., cardiomyocyte-specific expression of a transgene).

In some aspects, the present disclosure provides viral or non-viral vectors comprising expression cassettes encoding gene products, and methods of using the same. In some embodiments, the expression cassette described herein comprises a cardiac cell-specific promoter and/or enhancer (e.g., any of the cardiac cell-specific promoters and enhancers described herein, in any of the orientations as described herein). A polynucleotide encoding a gene product operably linked to the combination). In some embodiments, an expression cassette described herein consists of two copies of a polynucleotide encoding a gene product and a cardiac cell-specific promoter (e.g., any combination of those sequences, in either of the orientations as described herein) ) includes. In some embodiments, the expression cassette described herein comprises a cardiac cell-specific promoter and/or enhancer (e.g., a cardiac cell-specific promoter described herein, either one promoter, or an orientation as described herein. and one or two copies of a polynucleotide, a WPRE sequence, and/or a polyA sequence (e.g., in any of the orientations described herein) encoding a gene product operably linked to a , any combination of those sequences). In some embodiments, the expression cassettes described herein comprise a polynucleotide encoding a gene product operably linked to a cardiac cell-specific promoter and/or intron. In some embodiments, an expression cassette described herein comprises 1 or 2 copies of a polynucleotide encoding a gene product, 1 or 2 copies of a cardiac cell-specific promoter, 1 or 2 copies of a cardiac-specific enhancer. or more copies, and/or one or more intron sequences (e.g., in any combination of such sequences, in any of the orientations as described herein). In some embodiments, an expression cassette described herein comprises 1 or 2 copies of a polynucleotide encoding a gene product, 1 or 2 copies of a cardiac cell-specific promoter, 1 or 2 copies of a cardiac-specific enhancer. or more copies, one or more copies of one or more intron sequences (e.g., in any combination of such sequences, in any of the orientations as described herein), a WPRE sequence, and one or two copies of a polyA sequence (e.g., (or any combination of the corresponding sequences) in any of the orientations as described in In some embodiments, vectors comprising expression cassettes described herein can transduce, for example, cardiac cells. In some embodiments, the targeted cardiac cells express the gene product, e.g., provide high levels of expression of the gene product. In some aspects, the present disclosure provides pharmaceutical compositions comprising the vectors described herein. In some aspects, the present disclosure provides methods of treating a subject diagnosed with or at risk of heart disease (e.g., cardiomyopathy) using the vectors and pharmaceutical compositions of the present disclosure.

In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) virions as vectors for the expression cassettes described herein.

Abnormal calcium handling is a common feature of cardiomyopathy, and reduced Sarco/endoplasmic reticulum calcium ATPase (SERCA) activity plays a central role in both the initiation and progression of the disease. SERCA is a calcium pump that promotes the uptake, maintenance, and circulation of Ca ²⁺ ions in cardiac cells such as cardiomyocytes. SERCA activity is regulated by the inhibitory peptide phospholamban. There is considerable interest in increasing the activity of SERCA by increasing the abundance of a polypeptide called Dwarf Open Reading Frame (DWORF), which enhances SERCA activity through direct displacement of the SERCA inhibitory peptide phospholamban. Contacting SERCA with DWORF is a strategy to increase SERCA activity in cells.

In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) virions comprising polynucleotides encoding DWORF polypeptides or functional variants thereof, and methods of using the same. In some embodiments, rAAV virions described herein contain a cardiac cell-specific promoter and/or enhancer (e.g., any of the cardiac cell-specific promoters and enhancers described herein, in any of the orientations as described herein). A polynucleotide encoding a DWORF polypeptide or a functional variant thereof operably linked to a combination of ). In some embodiments, rAAV virions described herein contain one or two copies of a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, a WPRE sequence, and one or two copies of a polyA sequence (e.g., any combination of such sequences in either of the orientations as described herein. In some embodiments, the rAAV virions described herein comprise a polynucleotide encoding a DWORF polypeptide operably linked to a cardiac cell-specific promoter and/or intron. In some embodiments, the rAAV virions described herein have one or two copies of a polynucleotide encoding DWORF, one or two copies of a cardiac cell-specific promoter, one or two copies of a cardiac-specific enhancer. or more copies, and/or one or more intron sequences (e.g., in any combination of such sequences, in any of the orientations as described herein). In some embodiments, the rAAV virions described herein have one or two copies of a polynucleotide encoding DWORF, one or two copies of a cardiac cell-specific promoter, one or two copies of a cardiac-specific enhancer. or more copies, one or more copies of one or more intron sequences (e.g., in any combination of such sequences, in any of the orientations as described herein), a WPRE sequence, and one or two copies of a polyA sequence (e.g., (or any combination of the corresponding sequences) in any of the orientations as described in In some embodiments, rAAV virions described herein are capable of transducing cardiac cells into the host cell genome, for example, with a polynucleotide having a sequence encoding a DWORF polypeptide operably linked to a cardiac cell-specific promoter region. You can. In some embodiments, the targeted cardiac cells can express DWORF polypeptide and increase SERCA activity. Also provided in the present disclosure are pharmaceutical compositions comprising the rAAV virions described herein. In one aspect, the present disclosure provides a method of treating a subject diagnosed with or at risk of heart disease using the rAAV virions and pharmaceutical compositions of the present disclosure.

Terms

Unless the context indicates otherwise, the features of the invention may be used in any combination. Any feature or combination of features presented may be excluded or omitted. Additionally, certain features of the invention that are described in separate embodiments may be provided in combination in a single embodiment. Additionally, features of the invention described in a single embodiment may be provided individually or in any suitable sub-combination.

In general, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. The detailed description is divided into sections solely for the convenience of the reader, and the disclosure in any section may be combined with the disclosure in other sections.

Unless otherwise specified, practice of the present disclosure will utilize conventional techniques of tissue culture, immunology, molecular biology, cell biology, and recombinant DNA, which are within the scope of the art. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and Sell (2013) Stem Cells Handbook.

“And/or” means both “and” and “or,” and a list joined by “and/or” includes all possible combinations of one or more listed items.

As used herein, the term “about” when used to modify a numerical value indicates that a deviation of up to 10% above or below the numerical value remains within the intended meaning of the recited value.

“AAV” is an abbreviation for Adeno-Associated Virus. This term includes all subtypes of AAV, both naturally occurring and recombinant forms, except where the subtype is indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus. “AAV” includes AAV or any subtype. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genome sequences of various serotypes of AAV, as well as the sequences of native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7 ), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9). A publication describing AAV is Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al (2004) Virology 33:375-383; International Patent Publication Nos. WO2018/222503A1, WO2012/145601A2, WO2000/028061A2, WO1999/61601A2, and WO1998/11244A2; US Patent Application Nos. 15/782,980 and 15/433,322; and U.S. Patent Nos. 10,036,016, 9,790,472, 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, No. 7,198,951, No. 7,105,345 , 6,962,815, 6,984,517, and 6,156,303.

“rAAV virion” refers to a viral particle comprising at least one viral capsid protein (e.g., VP1) and an encapsulated rAAV vector (or fragment thereof).

An “infectious” virion or viral particle is one that contains a sufficiently assembled viral capsid and is capable of delivering polynucleotide components into cells to which the virion is tropic.

“Packaging” refers to the series of intracellular events that result in the assembly of rAAV virions, including encapsulation of the rAAV vector. The AAV “ rep ” and “ cap ” genes refer to polynucleotide sequences that encode replication and encapsulation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.” Packaging requires helper virus functions supplied by the helper virus itself or, more commonly, in recombinant systems, by a helper-free system (i.e., one or more helper plasmids). “Helper virus” for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to replicate and be packaged by mammalian cells. The helper virus may be an adenovirus, a herpesvirus, or a chickenpox virus such as cowpox.

As used herein, the term “inverted terminal repeat” or “ITR” refers to AAV viral cis-elements, so named because of their symmetry. These elements are essential for efficient propagation of the AAV genome. In some embodiments, the minimal elements essential for ITR function are believed to be a Rep-binding site and a terminal cleavage site plus a variable palindromic sequence that allows hairpin formation.

The term “parent capsid” or “parent sequence” refers to the reference sequence from which the particle capsid or sequence is derived. Unless otherwise specified, parent sequence refers to the sequence of a wild-type capsid protein of the same serotype as the engineered capsid protein.

“Recombinant,” as applied to a polynucleotide, means that the polynucleotide is distinct from polynucleotides found in nature, such that, for example, the polynucleotide is the product of various combinations of replication, restriction or ligation steps, and other procedures, or It means that the nucleotides were assembled from synthetic oligonucleotides. A “recombinant” protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that contains a recombinant polynucleotide and/or a recombinant protein, such as a recombinant capsid protein.

As used herein, the term "percent sequence identity", and the term "identity", when used to refer to percent sequence identity, refer to a reference nucleic acid after aligning sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. or for an amino acid sequence, the percentage of nucleic acid bases or amino acid residues in the candidate sequence that are identical to nucleic acid bases or amino acid residues in the reference sequence, respectively. Methods for sequence alignment are known in the art. Sequences can be aligned using various computer programs such as BLAST, available at ncbi.nlm.nih.gov. Alignment can be accomplished using publicly available computer software such as BLASTp, BLASTn, BLAST-2, ALIGN or MegAlign Pro (DNASTAR) software. For other techniques for alignment, see Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996); and Meth. Mol. Biol. 70: 173-187 (1997); J. Mol. Biol. Described in Method 48:44. One skilled in the art can select an appropriate alignment method depending on a variety of factors, including sequence length, divergence, and the presence of insertions or deletions relative to the reference sequence.

The terms “operably linked” and “operably linked” refer to a nucleic acid sequence placed in a functional relationship with another nucleic acid sequence. As used herein, these terms have meanings commonly known in the art. For example, a promoter is operably linked to a gene when the promoter is placed in a position that allows the promoter to initiate transcription of that gene. An enhancer is operably linked to a gene if, when bound by an appropriate transcription factor, the enhancer can modulate (e.g., enhance) the expression of that gene.

“Treatment,” “treating,” and “treat” refer to the use of an agent to reduce or alleviate the harmful or any other undesirable effects of a disease, disorder, or condition and/or symptoms thereof. Or it is defined as acting on a condition.

As used herein, terms such as “effective amount” or the like regarding amounts of a composition refer to an amount sufficient to induce a desired physiological result (e.g., treatment of a disease). An effective amount may be administered in one or more administrations, applications, or dosages. Such delivery depends on a number of variables, including the period over which the individual dosage unit will be used, the bioavailability of the composition, the route of administration, etc. However, the specific amount of composition (e.g., rAAV virion) for any particular subject will depend on the activity of the particular agent used, the subject's age, weight, general health, gender, and diet, time of administration, rate of excretion, combination of compositions, and other factors. It should be understood that this will depend on a variety of factors, including the severity of the particular condition being treated and the form of administration.

The phrase “pharmaceutically acceptable” means a reasonable benefit/risk that, within the scope of sound medical judgment, is suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction, or other problems or complications. Used herein to refer to compounds, materials, compositions, and/or dosage forms that correspond to ratios.

The terms “individual,” “subject,” and “patient” are used interchangeably herein and include, but are not limited to: human and non-human primates (e.g., simian monkeys); mammalian sports animals (eg, horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian companion animals (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

As used herein, “cardiomyopathy” refers to any disease or dysfunction that directly affects the myocardium. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibrogenic, hematological, genetic, or of unknown origin. Two basic types are recognized (1) the primary type, which consists of heart muscle disease of unknown etiology; and (2) secondary types, which consist of myocardial disease of known etiology or are associated with disease involving other organ systems. “Specific cardiomyopathy” refers to a heart disease that is associated with a specific systemic or cardiac disorder, examples of which include hypertensive and metabolic cardiomyopathy. Cardiomypathies include: dilated cardiomyopathy (DCM), a disorder in which left and/or right ventricular systolic pumping function is impaired, resulting in progressive cardiac hypertrophy; Hypertrophic cardiomyopathy, characterized by left ventricular hypertrophy without an obvious cause such as hypertension or aortic stenosis; and restrictive cardiomyopathy, characterized by abnormal diastolic function and excessively rigid ventricular walls that prevent ventricular filling. Cardiomyopathies also include left ventricular hypersystole, arrhythmogenic right ventricular cardiomyopathy, and arrhythmogenic right ventricular dysplasia.

“Heart failure” refers to a pathological condition in which abnormalities in cardiac function fail to pump blood at a rate commensurate with the requirements of the metabolic tissue and/or allow the heart to pump blood only from an abnormally elevated diastolic volume . Heart failure includes systolic and diastolic dysfunction. Patients with heart failure are those with low cardiac output (commonly secondary to ischemic heart disease, hypertension, dilated cardiomyopathy, and/or valvular or pericardial disease) and those with high cardiac output (commonly due to hyperthyroidism, anemia, pregnancy, arteriovenous fistula). , caused by beriberi and Paget's disease). Heart failure includes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF).

As used herein, the term “therapeutic gene” refers to a gene that, when expressed, provides a beneficial effect to the cell or tissue in which it resides, or to the mammal in which the gene is expressed. Examples of beneficial effects include alleviating signs or symptoms of a condition or disease, preventing or inhibiting a condition or disease, or imparting desired properties. Therapeutic genes include genes that partially or fully correct a genetic deficiency in a cell or mammal.

As used herein, the term “cardiac cell” refers to any cell present in the heart that provides cardiac functions, such as cardiac contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells, as used herein, include cells present in the epicardium, myocardium, or endocardium of the heart. Cardiac cells also include cells of cardiovascular structures, such as, for example, heart muscle cells or cardiomyocytes, and cells of coronary arteries or veins. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conduction cells, and cardiac pacemaker cells that make up the heart muscle, blood vessels, and cardiac cell support structures. Cardiac cells can be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

expression cassette

Expression Cassette Overview

Vectors of the present disclosure may include any of the expression cassettes described herein. In some embodiments, rAAV virions of the present disclosure comprise a viral genome comprising an expression cassette as shown in Figure 1 , Figure 7A-7C , or a variant thereof. The expression cassette may include a polynucleotide encoding any one of the gene products described herein, or a functional variant thereof, optionally operably linked to a promoter, optionally an intron, optionally a polyadenylation (poly(A)) signal, optionally a woodchuck hepatitis virus post-transcriptional element (WPRE), and optionally a transcription termination signal. The expression cassette may be flanked by inverted terminal repeats (ITRs). These components provide the ability for host cells to express the transgene after targeting, for example, by rAAV virions. The promoter sequence, when present, regulates the expression of the polynucleotide encoding the gene product.

The expression cassette comprises a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, optionally operably linked to a promoter, optionally an intron, optionally a polyadenylation (poly(A)) signal, optionally a woodchuck hepatitis virus transcript. post element (WPRE), and optionally a transcription termination signal. The promoter sequence, if present, regulates the expression of the polynucleotide encoding the DWORF polypeptide or functional variant thereof. The promoter sequence may be a cardiac cell-specific promoter. The promoter sequence may additionally be operably linked to an enhancer, such as any of the cardiac cell specific enhancers described herein.

In any of the constructs shown in Figures 1 and 7A-7C , the DWORF nucleotide sequence can be replaced with a nucleotide sequence encoding another gene product or polypeptide, such as any of the gene products or polypeptides described herein ( For example, see descriptions of transgenes and gene products encoded by these transgenes below). Accordingly, in some embodiments, provided herein is any one of the expression cassettes shown in Figures 1 and 7A-7C , wherein the DWORF nucleotide sequence is replaced with a nucleotide sequence encoding another gene product or polypeptide.

Additionally, in any of the expression cassettes set forth in Table 1, the DWORF nucleotide sequence may be replaced with a nucleotide sequence encoding another gene product or polypeptide, such as any of the gene products or polypeptides described herein (e.g., See description below for transgenes and gene products encoded by these transgenes). Additionally, in any of the expression cassettes shown in Table 1, the ITR sequence may be omitted. Accordingly, in some embodiments, provided herein is any one of the expression cassettes set forth in Table 1, wherein the DWORF nucleotide sequence is replaced with a nucleotide sequence encoding another gene product or polypeptide and/or the specific ITR sequence is absent. No.

transgene

In some embodiments, the expression cassette of the present disclosure comprises a transgene. The transgene may include a nucleotide sequence encoding any polypeptide for use in treating or preventing, or alleviating the symptoms of, a heart disease or disorder. Promoters, enhancers and combinations thereof described herein are operably linked to transgenes encoding the products. A transgene may be a gene or a nucleotide sequence that encodes a product or functional fragment thereof. The product may be, for example, a polypeptide or a non-coding nucleotide. Non-coding nucleotides mean that sequences transcribed from a transgene or nucleotide sequence are not translated into a polypeptide. In some embodiments, the product encoded by a transgene or nucleotide operably linked to an enhancer described herein is a non-coding polynucleotide. The non-coding polynucleotide may be RNA, such as, for example, microRNA (miRNA or mIR), short hairpin RNA (shRNA), long non-coding RNA (lnRNA), and/or short interfering RNA (siRNA). In some embodiments, the transgene encodes a product that is natively expressed by heart cells, such as cardiomyocytes. In some embodiments, the transgene encodes a product that is natively expressed in cell types other than cardiac cells. Without limitation, cell types other than cardiac fibroblasts may be derived from any multicellular organism, unicellular organism, or microorganism.

In some embodiments, the transgene encodes a polypeptide. In some embodiments, the transgene encodes a non-coding polynucleotide, such as, for example, a microRNA (miRNA or mIR).

In some embodiments, the transgene is a growth factor such as cadherin, connexin, Cx43, fibroblast growth factor (FGF)-2, and transforming growth factor-β, and a family of growth factors such as interleukin (IL)-1β and IL-6. Cytokines, leukemia inhibitory factor, cardiotrophin-1, cardiogenic transcription factor, insulin-like growth factor, GATA4, MEF2C, TBX5, ESRRG, MESP1, MYOCD, ZFPM2, HAND2, miR-1, miR-133, Oct4, Contains a sequence encoding a product selected from Sox2, Klf4, c-Myc, SRF, SMARCD3, Nkx2-5, Akt, PKB, Baf60c, BMP4, miR-208, and miR-499.

In some embodiments, the transgene encodes a functional cardiac protein. In some embodiments, the gene product is a genome-editing endonuclease (optionally with a guide RNA, single guide RNA, and/or repair template) that replaces or repairs a non-functional cardiac protein with a functional cardiac protein. Functional cardiac proteins include, but are not limited to: cardiac troponin T; cardiac myofibrillar protein; β-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac a-actin; a-tropomyosin; cardiac troponin I; cardiac myosin-binding protein C; 4.5 LIM protein 1; titin; 5'-AMP-activated protein kinase subunit gamma-2; Troponin I type 3, myosin light chain 2, actin alpha cardiac muscle 1; cardiac LIM protein; caveolin 3 (CAV3); galactosidase alpha (GLA); Lysosome-Associated Membrane Protein 2 (LAMP2); mitochondrial transfer RNA glycine (MTTG); mitochondrial transfer RNA isoleucine (MTTI); mitochondrial transfer RNA lysine (MTTK); mitochondrial transfer RNA glutamine (MTTQ); myosin light chain 3 (MYL3); Troponin C (TNNC1); transthyretin (TTR); Sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a); stroma-derived factor-1 (SDF-1); Adenylate cyclase-6 (AC6); Beta-ARKct (β-Adrenergic Receptor Kinase C Terminal); Fibroblast growth factor (FGF); platelet derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia inducible growth factor; thymosin beta 4 (TMSB4X); nitric oxide synthase-3 (NOS3); unocartin 3 (UCN3); Melusine; Apolipoprotein-E (ApoE); superoxide dismutase (SOD); and S100A1 (small calcium binding protein; see, e.g., Ritterhoff and Most (2012) Gene Ther. 19:613; Kraus et al. (2009) Mol. Cell. Cardiol. 47:445).

In some embodiments, the transgene can treat or prevent coronary heart disease. In some embodiments, the transgene is vascular endothelial growth factor (VEGF), VEGF isoform, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-D ^dNdC , VEGF-A _116A , VEGF-A ₁₆₅ , VEGF-A ₁₂₁ , VEGF-2, placental growth factor (PIGF), fibroblast growth factor 4 (FGF-4), human growth factor (HGF), human granulocyte colony-stimulating factor (hGCSF), and hypoxia-inducible factor 1α ( HIF-1α).

In some embodiments, the transgene can treat or prevent heart failure. In some embodiments, the transgene can treat or prevent chronic heart failure. In some embodiments, the transgene is SERCA2a, stromal cell derived factor-1 (SDF-1), adenylyl cyclase type 6, S100A1, miRNA-17-92, miR-302-367, anti-miR-29a antibody. -miR-30a, anti-miR-141, cyclin A2, cyclin-dependent kinase 2, Tbx20, miRNA-590, miRNA-199, antisense oligonucleotide against Lp(a), interfering RNA against PCSK9, apolipoprotein C -III, an antisense oligonucleotide for lipoprotein lipase ^S447X , an antisense oligonucleotide for apolipoprotein B, an antisense oligonucleotide for c-myc, and an E2F oligonucleotide decoy.

In some embodiments, the transgene encodes a gene product, the expression of which complements a defect in the gene responsible for the genetic disorder. The present disclosure provides polynucleotides encoding one or more of the following, for example, for use in the disorders indicated in the following parentheses, or for use in other disorders caused by each: TAZ (Barth ) syndrome); FXN (Freidrich's ataxia); CASQ2(CPVT); FBN1 (Marfan); RAF1 and SOS1 (Noonan); SCN5A (Brugada); KCNQ1 and KCNH2 (long QT syndrome); DMPK (myotonic dystrophy 1); LMNA (Land Muscle Dystrophy Type 1B); JUP (Naxos); TGFBR2 (Loeys-Dietz); EMD (X-linked EDMD); and ELN (SV aortic stenosis). In some embodiments, the polynucleotide encodes one or more of the following: cardiac troponin T (TNNT2); BAG family molecular chaperone regulator 3 (BAG3); myosin heavy chain (MYH7); Tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5'-AMP-activated protein kinase subunit gamma-2 (PRKAG2); Troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); Actin, alpha cardiac muscle 1 (ACTC1); Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

In some embodiments, the transgene is DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulator 3 (BAG3), phospholamban (PLN), alpha-crystallin B chain (CRYAB). ), LMNA (e.g. lamin A and lamin C isoforms), troponin I type 3 (TNNI3), lysosome-associated membrane protein 2 (LAMP2, e.g. LAMP2a, LAMP2b and LAMP2c isoforms), desmoplakin (DSP, e.g. DPI and DPII isoforms), desmoglein 2 (DSG2), and junctional plakoglobin (JUP). In some embodiments, the transgene comprises a nucleotide sequence encoding a human protein. In some embodiments, the transgene comprises a human nucleotide sequence (human DNA sequence). In some embodiments, the transgene comprises a codon optimized DNA sequence. In some embodiments, the transgene comprises a nucleotide sequence encoding a wild-type protein or a functionally active fragment thereof.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a DWORF polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a juntophilin 2 (JPH2) polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a full-length JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of a JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of a JPH2 polypeptide that retains JPH2 activity.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a BAG3 polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a CRYAB polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding an LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the lamin A isoform of LMNA. In some embodiments, the transgene comprises a polynucleotide sequence encoding the lamin C isoform of LMNA.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a TNNI3 polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a PLN polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2a isoform. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2b isoform. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2c isoform.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPI isoform of DSP. In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPII isoform of DSP.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSG2 polypeptide.

In some embodiments, the transgene comprises a polynucleotide sequence encoding a JUP polypeptide.

It will be understood that the transgenes described herein are not limiting, and that transgenes useful for treating heart disease may be developed for use in the expression cassettes described herein.

DWORF transgene

In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette provides for increased expression of the DWORF polypeptide in cardiac cells. In some embodiments, the heart cells are cardiomyocytes. In some embodiments, the expression of the DWORF polypeptide can be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of the DWORF polypeptide factor in an untreated subject. In some embodiments, expression of the DWORF polypeptide can be increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of the DWORF polypeptide in an untreated subject. In some embodiments, the DWORF polypeptide may be expressed at any detectable level in cardiac cells, while the DWORF polypeptide may be expressed at no or undetectable levels in untreated subjects. Stated another way, cardiac cells administered rAAV virions may express the DWORF polypeptide at a higher abundance than cardiac cells that have only endogenous (i.e. native) expression of the DWORF polypeptide.

DWORF polypeptide is an endogenous enhancer of SERCA calcium pump activity, which is a desirable drug target for modulation of cardiac contractility. DWORF is also an unusually small protein, which makes it a good candidate for delivery to target cells or tissues by rAAV virions. Because DWORF is an endogenous protein, expression of DWORF in humans is not immunogenic, allowing long-term administration and expression. The structural characteristics of DWORF polypeptide are as follows. Preferentially, the polypeptide may have 5 to 35 consecutive residues of the dwarf open reading frame (DWORF) located on chromosome 3 of mammalian species, including mice and humans (Nelson et al . Science. 351: 271-275 (2016 ); see US Pat. No. 10,570,183). Accordingly, although including the term “comprising,” the term “peptide having up to Typically, the peptide will be no more than 35 residues, which in turn includes a DWORF of no more than 20 consecutive residues. The total length is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. , 29, 30, 31, 32, 33, 34, or 35 residues. 5-34/35 residues, 6-34/35 residues, 7-50 residues, 7-25 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 A range of peptide lengths of up to 20 residues is considered. The number of consecutive DWORF residues can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. 5-20 residues, 5-20 residues, 6-20 residues, 7-20 residues and 5-15 residues, 5-15 residues, 6-15 residues, or consecutive residues of 7-15 residues range is considered. Exemplary DWORF sequences can be found in Table 2a.

In some embodiments, the DWORF polypeptide is a human DWORF polypeptide. In some embodiments, the expression cassette comprises a single polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide. In some embodiments, the polynucleotide sequence encoding DWORF is codon optimized. In some embodiments, the DWORF polypeptide comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the DWORF polypeptide comprises a polynucleotide sequence that shares at least 98% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the DWORF polypeptide comprises the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

Other Exemplary Transgenes

In some embodiments, the expression cassette of the present disclosure is a gene product (not a DWORF), such as JPH2, BAG3, CRYAB, LMNA (e.g., lamin A or lamin C isoform), TNNI3, PLN, LAMP2 ( poly, which encodes a polypeptide selected from, for example, LAMP2a, LAMP2b, or LAMP2c isoforms), DSP (e.g., DPI or DPII isoform), desmoglein 2 (DSG2), and junctional plakoglobin (JUP). Contains a nucleotide sequence. In some embodiments, the expression cassette provides for increased expression of the gene product in cardiac cells. In some embodiments, the heart cells are cardiomyocytes. In some embodiments, expression of a polypeptide encoded by a polynucleotide sequence can be increased by 5%, 10%, 15%, 20%, or 25% compared to expression in an untreated subject. In some embodiments, expression of a polypeptide encoded by a polynucleotide sequence can be increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to expression in an untreated subject. In some embodiments, the polypeptide encoded by the polynucleotide sequence may be expressed at any detectable level in cardiac cells, whereas it may be expressed at no or undetectable levels in untreated subjects. In some embodiments, cardiac cells to which a vector described herein is administered are capable of expressing the polypeptide encoded by the polynucleotide sequence at a higher abundance than in cardiac cells that have only endogenous (i.e., native) expression of the polypeptide.

In some embodiments, the polypeptide is a human polypeptide. In some embodiments, the polynucleotide sequence encoding the polypeptide is codon optimized. In some embodiments, the expression cassette comprises a single polynucleotide sequence encoding a polypeptide. In some embodiments, the expression cassette includes two polynucleotide sequences encoding a polypeptide. In some embodiments, the expression cassette comprises two polynucleotide sequences encoding polypeptides, wherein at least one of the sequences is codon optimized.

In some embodiments, the polynucleotide sequence encodes JPH2, e.g., human JPH2. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:201. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding JPH2, e.g., human JPH2. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is JPH2, e.g., human JPH2. In some embodiments, the JPH2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:202.

In some embodiments, the polynucleotide sequence encodes an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the polynucleotide sequence of the N-terminal fragment of JPH2 has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:227. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the N-terminal fragment of the JPH2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:228. The human sequence of the N-terminal fragment of JPH2 corresponds to the mouse JPH2 N-terminal peptide with amino acids 1-565 generated by calpain cleavage (see Guo et al., 2018, Science 362, doi: 10.1126/science.aan3303) .

In some embodiments, the polynucleotide sequence encodes BAG3, e.g., human BAG3. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:203. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding BAG3, e.g., human BAG3. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is BAG3, e.g., human BAG3. In some embodiments, the BAG3 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:204.

In some embodiments, the polynucleotide sequence encodes CRYAB, e.g., human CRYAB. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:205. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding CRYAB, e.g., human CRYAB. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is CRYAB, e.g., human CRYAB. In some embodiments, the CRYAB polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:206.

In some embodiments, the polynucleotide sequence encodes an LMNA, such as human LMNA. In some embodiments, the polynucleotide sequence encodes a lamin A isoform of LMNA, e.g., human lamin A. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:207. In some embodiments, the polynucleotide sequence encodes a lamin C isoform of LMNA, e.g., human lamin C. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:209. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding an LMNA polypeptide. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding lamin A or lamin C, e.g., human lamin A or lamin C. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is LMNA, eg, human LMNA. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a LMNA, e.g., the lamin A isoform of human LMNA. In some embodiments, the lamin A polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:208. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is an LMNA, e.g., the lamin C isoform of human LMNA. In some embodiments, the lamin C polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:210.

In some embodiments, the polynucleotide sequence encodes TNNI3, e.g., human TNNI3. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:211. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding TNNI3, e.g., human TNNI3. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is TNNI3, e.g., human TNNI3. In some embodiments, the TNNI3 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:212.

In some embodiments, the polynucleotide sequence encodes a PLN, such as a human PLN. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:229. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding a PLN, eg, a human PLN. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is PLN, eg, human PLN. In some embodiments, the PLN polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:230.

In some embodiments, the polynucleotide sequence encodes LAMP2, e.g., human LAMP2. In some embodiments, the polynucleotide sequence encodes a LAMP2a isoform of LAMP2, e.g., human LAMP2a. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:213. In some embodiments, the polynucleotide sequence encodes a LAMP2b isoform of LAMP2, e.g., human LAMP2b. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:215. In some embodiments, the polynucleotide sequence encodes a LAMP2c isoform of LAMP2, e.g., human LAMP2c. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:217. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding LAMP2, e.g., human LAMP2. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding LAMP2a, LAMP2b, or LAMP2c. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a LAMP2 polypeptide, e.g., human LAMP2. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is LAMP2, e.g., the LAMP2a isoform of human LMNA2. In some embodiments, the LAMP2a polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:214. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is LAMP2, e.g., the LAMP2b isoform of human LMNA2. In some embodiments, the LAMP2b polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:216. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is LAMP2, e.g., the LAMP2c isoform of human LMNA2. In some embodiments, the LAMP2c polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:218.

In some embodiments, the polynucleotide sequence encodes a DSP, such as a human DSP. In some embodiments, the polynucleotide sequence encodes a DSP, e.g., a DPI isoform of human DPI. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:219. In some embodiments, the polynucleotide sequence encodes a DPII isoform of DSP, such as human DPII. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:221. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding a DSP polypeptide. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding DPI or DPII, for example human DPI or DPII. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a DSP, eg, a human DSP. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a DSP, e.g., the DPI isoform of human DSP. In some embodiments, the DPI polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:220. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a DSP, e.g., the DPII isoform of human DSP. In some embodiments, the DPII polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:222.

In some embodiments, the polynucleotide sequence encodes DSG2, such as human DSG2. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:223. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding DSG2, e.g., human DSG2. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is DSG2, e.g., human DSG2. In some embodiments, the DSG2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:224.

In some embodiments, the polynucleotide sequence encodes a JUP, e.g., a human JUP. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:225. In some embodiments, the polynucleotide sequence is a codon optimized sequence encoding a JUP, e.g., a human JUP. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is a JUP, eg, a human JUP. In some embodiments, the JUP polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:226.

In some embodiments, any one of the other polynucleotide sequences described herein can be used in any of the expression constructs described herein. In some embodiments, such polynucleotide sequence encodes any of the gene products or polypeptides described herein. The polynucleotide sequence may be sequence optimized (e.g., for expression in humans). In some embodiments, the sequence encodes a human polypeptide. The sequences of the polynucleotides and polypeptides described herein are known in the art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with such sequences are also contemplated herein. Exemplary sequences can be found in Table 2b.

Promoters and Enhancers

In some embodiments, the expression cassette of the present disclosure includes a promoter. As used herein, the term “promoter” refers to a DNA sequence that promotes RNA synthesis by inducing the binding of RNA polymerase. Promoter and corresponding protein or polypeptide expression can be ubiquitous, meaning that they are potently active in a wide range of cells, tissues and species or cell type specific, tissue specific, or species specific. Examples of ubiquitous promoters include the CAG promoter and the CMB promoter (Yue et al. BioTechniques 33:672-678 (2002)). A promoter may be “constitutive,” i.e., continuously active, or “inducible,” meaning that the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Additionally, enhancer sequences, which may or may not be adjacent to the promoter sequence, are included in the nucleic acid constructs or vectors of the invention. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

A variety of promoters can be used. Promoters can be cell type specific. Constitutive promoters are used in expression cassettes, such as the chicken β-actin promoter (CAG), the simian virus 40 (SV40) promoter, and the cytomegalovirus enhancer fused to the herpes simplex virus thymidine kinase (HSV-TK) promoter. (Damdindorj et al., PLoS One . 9:e106472 (2014)). Other cell type specific promoters may also be used. Cardiac cell specific promoters include, for example, the MLC2v promoter (Phillips et al., Hypertension . 39:651-5 (2002)) and the cardiac troponin-T (cTnT) promoter (Konkalmatt et al., Circ Cardiovasc Imaging . 6:478- 486 (2013)). The transgene polynucleotide sequence within the expression cassette may be, for example, an open reading frame encoding a protein. The ITR within the expression cassette functions as a marker used in viral packaging of the expression cassette (Clark et al., Hum Gene Ther. 6:1329-41 (1995)).

Advantageously, the promoter, optionally together with an enhancer, allows expression of a polynucleotide encoding a polypeptide (e.g. a DWORF polypeptide) or a functional variant thereof in a target cell.

In some embodiments, the expression cassette includes a single promoter. In some embodiments, the expression cassette includes at least one promoter. In some embodiments, the expression cassette includes two promoters. In some embodiments, the expression cassette includes a ubiquitous promoter. In some embodiments, the expression cassette includes an inducible promoter. In some embodiments, the expression cassette includes a cell type specific promoter. In some embodiments, a promoter specifically promotes expression of a polynucleotide encoding a polypeptide or functional variant thereof in cardiac cells (e.g., cardiomyocytes). In some embodiments, the promoter specifically promotes expression of a polynucleotide encoding a DWORF polypeptide or functional variant thereof in cardiac cells. In some embodiments, the promoter specifically promotes expression of a polynucleotide encoding a DWORF polypeptide or a functional variant thereof in cardiomyocytes. Table 3 provides exemplary promoter and enhancer sequences.

In some embodiments, the promoter is the chicken cardiac troponin T (cTnT or ccTnT) promoter. In some embodiments, the chicken cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11.

In some embodiments, the promoter is the human cTnT promoter. In some embodiments, the promoter is the short human cTnT promoter. In some embodiments, the short human cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12. In some embodiments, the short human cTnT promoter comprises SEQ ID NO: 12. In some embodiments, the promoter is the long human cTnT promoter. In some embodiments, the long human cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 13. In some embodiments, the long human cTnT promoter comprises SEQ ID NO: 13.

An expression cassette may include one or more enhancers. As used herein, the term “enhancer” refers to a DNA sequence that promotes RNA synthesis by inducing the binding of a transcriptional regulatory protein (e.g., transcription machinery) and RNA polymerase. An enhancer can be operably linked to a promoter and can regulate expression of a transgene operably linked to the promoter. The presence of an enhancer can modulate transgene expression, for example by increasing or decreasing expression. Enhancers can modulate transgene expression, for example by increasing expression levels in desired cell types, such as cardiac cells. Enhancers can modulate transgene expression, for example, by reducing expression levels in “off-target” cell types, or cell types for which expression is undesirable.

In some embodiments, the expression cassette includes a single enhancer. In some embodiments, the expression cassette includes at least one enhancer. In some embodiments, the expression cassette includes two enhancers. In some embodiments, the expression cassette includes three enhancers. In some embodiments, the expression cassette includes four enhancers. In some embodiments, the expression cassette includes an enhancer operably linked to a promoter. For example, the ACTC1 cardiac enhancer can be linked to the human cTnT promoter. In some embodiments, the expression cassette includes an enhancer operably linked to another enhancer. For example, the ACTC1 cardiac enhancer can be operably linked to an αMHC enhancer. In some embodiments, the expression cassette includes an enhancer operably linked to a promoter and operably linked to another enhancer.

In some embodiments, the enhancer comprises ACTC1 cardiac enhancer (ACTC1e). In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO:78. In some embodiments, the enhancer comprises an αMHC enhancer (αMHCe). In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. In some embodiments, the αMHC enhancer comprises SEQ ID NO:79.

intron

Expression cassettes may include intronic sequences, such as synthetic or chimeric intronic sequences. Intronic sequences can be used to control the length (i.e. size) of the expression cassette to improve recombinant AAV packaging. Intron sequences can be used to improve the efficiency of transgene expression (i.e., mRNA production or transcription) in host cells containing the expression cassette. In some embodiments, the expression cassette includes an intron. In some embodiments, the intron includes a CMV intron (CMVint). In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:80. In some embodiments, the CMV intron includes SEQ ID NO:80. In some embodiments, the intron includes a chimeric intron. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:81. In some embodiments, the chimeric intron comprises SEQ ID NO:81.

WPRE sequences and other post-transcriptional elements

In some embodiments, the expression cassette includes post-transcriptional regulatory elements.

In some embodiments, the expression cassette includes a woodchuck hepatitis virus post-transcriptional element (WPRE). The WPRE sequence can be inserted proximal to the 3' end of the transgene, for example in a viral vector, to optimize gene expression in the viral vector (Lee et al. Exp Physiol. 90:33-37 2005)). In some embodiments, the WPRE comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:26. In some embodiments, the WPRE comprises SEQ ID NO:26.

polyadenylation sequence

In some embodiments, the expression cassette includes a poly(A) signal sequence. In some embodiments, the poly(A) signal is a BGH poly(A) sequence. In some embodiments, the BGH poly(A) sequence comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:27. In some embodiments, the poly(A) signal is an SV40 poly(A) signal. In some embodiments, the SV40 poly(A) sequence comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:28.

inverted terminal repeat sequence

In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITR). In some embodiments, the ITR comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14, and/or SEQ ID NO: 15. do.

Exemplary Expression Cassettes

The present disclosure provides expression cassettes comprising polynucleotides comprising the 5' to 3' arrangement (sometimes referred to as orientation) of the elements. In some embodiments, the elements include one or more promoters; Optionally one or more enhancers; optionally one or more introns; One or more transgenes; Optionally one or more WPRE sequences; and optionally one or more polyadenylation sequences (p(A)). Exemplary sequences of elements within a polynucleotide are shown in Figures 1, 7A, 7B, 7C, and Table 1. Additionally, exemplary orientations of elements relative to polynucleotides are presented in Figures 1, 7A, 7B, 7C, and Table 1. In some embodiments, the 5' to 3' arrangement of the elements is selected from:

5'-promoter-transgene-WPRE-p(A)-3';

5'-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A);

5'-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-Enhancer-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';

5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

5'-Promoter-Intron-Transgene-WPRE-p(A)-Promoter-Intron-Transgene-p(A)-3'; and

5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

In some embodiments, the expression cassette described herein comprises a second nucleotide comprising an array of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. Achieve increased expression levels of the transgene compared to the expression cassette. In some embodiments, the level of expression is increased by about 1.5-fold to about 150-fold compared to the second expression cassette.

In some embodiments, the expression cassettes provided herein include the following elements (elements may be described herein, e.g., sequences thereof are provided herein):

5'-promoter-transgene-WPRE-p(A)-3';

5'-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A);

5'-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-Enhancer-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';

5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';

5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

5'-Promoter-Intron-Transgene-WPRE-p(A)-Promoter-Intron-Transgene-p(A)-3'; or

5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

In the expression cassettes described herein (e.g., those listed above), the orientation of promoters, enhancers, transgenes, and poly(A) elements (e.g., more than one promoter, one promoter, optionally an enhancer, and operative The operably linked transgene may be oriented in the forward direction (whereas the other promoter, optionally the enhancer and the operably linked transgene may be oriented in the reverse direction).

In some embodiments, the expression cassette provided herein includes the following elements:

5'-Heart-specific promoter-transgene-WPRE-p(A)-3';

5'-cardiac-specific promoter-intron (eg, chimeric intron)-transgene-WPRE-p(A)-3';

5'-cardiac-specific promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A), wherein both the promoter and transgene sequences are in the same forward orientation;

5'-cardiac-specific promoter-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-cardiac-specific promoter-intron (eg, CMV intron)-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, ACTC1e)-cardiac-specific promoter-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, αMHCe)-cardiac-specific promoter-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, ACTC1e)-cardiac-specific promoter-intron (eg, CMV intron)-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, αMHCe)-cardiac-specific promoter-intron (eg, CMV intron)-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, ACTC1e)-Enhancer (eg, αMHCe)-cardiac-specific promoter-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (eg, αMHCe)-Enhancer (eg, ACTC1e)-cardiac-specific promoter-transgene-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (e.g. ACTC1e)-Enhancer (e.g. αMHCe)-cardiac-specific promoter-intron (e.g. CMV intron)-transgene-WPRE-p(A) (e.g. , bGHpA)-3';

5'-Enhancer (e.g., αMHce)-Enhancer (e.g., ACTC1e)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g. , bGHpA)-3';

5'-cardiac-specific promoter-transgene with codon optimized polynucleotide sequence-WPRE-p(A) (eg, bGHpA)-3';

5'-Enhancer (e.g., αMHCe)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-p(A)( For example, SV40pA)-transgene (e.g., with a codon optimized polynucleotide sequence)-intron (e.g., chimeric intron)-cardiac-specific promoter-enhancer (e.g., ACTC1e)-3 '(optionally, wherein the first transgene in the sequence and the promoter/enhancer sequence operably linked thereto are in the forward orientation and the second transgene in the sequence and the promoter/enhancer sequence operably linked thereto are in the reverse orientation);

5'-Enhancer (e.g. αMHCe)-cardiac-specific promoter-intron (e.g. CMV intron)-transgene-WPRE-p(A) (e.g. bGHpA)-enhancer (e.g. , ACTC1e)-cardiac-specific promoter-intron (e.g., chimeric intron)-transgene (e.g., with codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)- 3' (optionally, wherein the first and second transgenes in sequence and promoter/enhancer sequences operably linked thereto are in forward orientation);

5'-p(A) (e.g., bGHpA)-WPRE-transgene-intron (e.g., CMV intron)-cardiac-specific promoter-enhancer (e.g., αMHCe)-enhancer (e.g. , ACTC1e)-cardiac-specific promoter-intron (e.g., chimeric intron)-transgene (e.g., with codon optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3 '(optionally, wherein the first transgene in the sequence and the promoter/enhancer sequence operably linked thereto are in a reverse orientation and the second transgene in the sequence and the promoter/enhancer sequence operably linked thereto are in the forward orientation);

5'-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-p(A) (e.g., SV40pA)-transgene (e.g., having a codon optimized polynucleotide sequence) -intron (e.g., chimeric intron) -cardiac-specific promoter-3' (optionally, wherein the first transgene in the sequence and operably thereto the linked promoter/enhancer sequence is in forward orientation and the second transgene in sequence and the promoter/enhancer sequence operably linked thereto are in reverse orientation;

5'-cardiac-specific promoter-intron (e.g., CMV intron)-WPRE-p(A) (e.g., bGHpA)-cardiac-specific promoter-intron (e.g., chimeric intron)-grafting Gene (e.g., having a codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3' (optionally, wherein the first and second transgenes in the sequence and operable thereto linked promoter/enhancer sequences are in forward orientation); or

5'-p(A) (e.g., bGHpA)-WPRE-transgene-intron (e.g., CMV intron)-cardiac-specific promoter-cardiac-specific promoter-intron (e.g., chimeric intron )-transgene (e.g., having a codon optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3'(optionally, wherein the first transgene of the sequence and operably the linked promoter/enhancer sequence is in reverse orientation and the second transgene in sequence and the promoter/enhancer sequence operably linked thereto are in forward orientation).

In the expression cassettes described herein (e.g., those described above), the cardiac specific promoter may be the short human cTnT promoter (e.g., hcTnTp) or the chicken cTnT promoter (e.g., ccTnTp). More specific examples of the expression cassettes described above can be found, for example, in Figures 1, 7A, 7B, 7C and Table 1.

In some embodiments, the expression cassette described herein is a second nucleotide comprising a polynucleotide having an arrangement of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. Allows for increased expression levels of the transgene compared to the expression cassette. In some embodiments, the level of expression is increased by about 1.5-fold to about 150-fold compared to the second expression cassette.

In some embodiments, one or more (e.g., 1, 2, 3 or 4) elements of the expression cassette described herein may be omitted.

In some embodiments, one or more (e.g., 1, 2, 3 or 4) elements of an expression cassette described herein may be replaced with another element, such as a functionally equivalent element.

In some embodiments of the expression cassettes provided herein, the WPRE element is replaced with any other post-transcriptional regulatory element known in the art. In some embodiments, expression cassettes provided herein include any post-transcriptional regulatory elements known in the art. In some embodiments, expression cassettes provided herein do not include post-transcriptional regulatory elements (e.g., do not include WPRE elements). In some embodiments, the expression cassette provided herein includes a WPRE.

In some embodiments of the expression cassettes provided herein, the bGHpA and/or SV40pA poly(A) element is replaced with any other poly(A) element known in the art. In some embodiments, expression cassettes provided herein include any poly(A) element known in the art. In some embodiments, the expression cassettes provided herein do not include a poly(A) element. In some embodiments, the expression cassette provided herein does not include bGHpA. In some embodiments, the expression cassette provided herein does not include SV40pA. In some embodiments, the expression cassette provided herein does not include bGHpA or SV40pA. In some embodiments, the expression cassette provided herein includes one or both of bGHpA and SV40pA.

In some embodiments of the expression cassettes provided herein, the CMV intron and/or chimeric intronic element is replaced with any other intronic element known in the art. In some embodiments, expression cassettes provided herein include any intronic element known in the art. In some embodiments, expression cassettes provided herein do not include introns. In some embodiments, the expression cassettes provided herein do not include a CMV intron. In some embodiments, the expression cassettes provided herein do not include chimeric introns (e.g., do not include Chim int). In some embodiments, the expression cassettes provided herein do not include a CMV intron or Chim int. In some embodiments, the expression cassette provided herein includes one or both a CMV intron and a Chim int.

It should be understood that exemplary orientations of expression cassettes may include inverted terminal repeat (ITR) sequences flanking the 5' and 3' ends of the expression cassette. It should be understood that ITR sequences may be optional. In some embodiments, the expression cassettes described herein do not include ITR sequences (e.g., non-AAV, such as DNA plasmid based expression cassettes).

Operably linked elements, such as those in the exemplary orientations described above, may be on one or both strands of the polynucleotide.

In some embodiments, the expression cassette includes one copy of the sequence encoding the polypeptide (i.e., one copy of the transgene). In some embodiments, the expression cassette includes two copies of the sequence encoding the polypeptide (i.e., two copies of the transgene). In some embodiments, when an expression cassette includes two copies of a sequence encoding a polypeptide, the two “copies” are not identical. Without being bound by any theory, the use of two sequences encoding non-identical polypeptides can prevent DNA recombination within the vector. In some embodiments, the expression cassette includes one copy with the original DNA sequence encoding the polypeptide and one copy with the codon-optimized DNA sequence encoding the polypeptide. In some embodiments, when an expression cassette comprises two copies of a sequence encoding a polypeptide, the two “copies” are identical.

In some embodiments, the expression cassette includes one or more promoters described herein (with or without one or more enhancers described herein) that drive one or more copies of a transgene (e.g., any one of the transgenes described herein). Includes. In some embodiments, the expression cassette does not include an enhancer (e.g., αMHCe and/or ACTC1e). In some embodiments, the expression cassette includes one or more enhancers, such as cardiac-specific enhancers (e.g., αMHCe and/or ACTC1e). In some embodiments, the expression cassette includes an αMHce enhancer (and, optionally, does not include an ACTC1e enhancer). In some embodiments, the expression cassette includes an ACTC1e enhancer (and, optionally, does not include an αMHce enhancer). In some embodiments, the expression cassette includes at least two enhancers in the order of first αMHCe and then ACTC1e. In some embodiments, the expression cassette includes at least two enhancers in the order of first ACTC1e and then αMHCe. In some embodiments, the expression cassette includes intronic elements, such as CMV intronic elements and/or chimeric introns (such as Chim introns described herein). In some embodiments, the expression cassette includes intronic elements but does not include an enhancer. In some embodiments, the expression cassette comprises intronic elements (e.g., CMV intron and/or chimeric intron) and further comprises an enhancer (e.g., αMHce and/or ACTC1e). In some embodiments, the expression cassette comprises a transgene with a codon optimized polynucleotide sequence. In some embodiments, the expression cassette includes a transgene with a codon optimized polynucleotide sequence, but does not include an enhancer. In some embodiments, the expression cassette comprises a transgene with a codon optimized polynucleotide sequence and further comprises an enhancer (e.g., αMHCe and/or ACTC1e). In some embodiments, the expression cassette is one, two or more cassettes described herein that drive expression of one or more promoters described herein and one or more copies of a transgene (with or without CMV intron or chimeric intronic elements). Enhancers (including, for example, αMHCe and/or ACTC1e enhancers). In some embodiments, the promoter is a cardiac-specific promoter, such as the human cTnT promoter (such as the short human promoter, hcTnTp) and/or the chicken cTnT promoter (such as ccTnTp). In some embodiments, the enhancer is a cardiac specific enhancer, such as αMHce and/or ACTC1e. In some embodiments, two or more cardiac-specific enhancers are used, where two or more of the enhancers may be the same or different (e.g., both or both αMHce, both or both ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardio-specific enhancers are used, where the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e) . In some embodiments, the transgene comprises a non-codon optimized polynucleotide sequence encoding the gene product. In some embodiments, the order of elements is as shown in any of the expression cassettes shown in Figures 1, 7A, 7B, 7C, and Table 1. For example, (i) the order of promoter and transgene elements can be as shown in Figure 1, Figure 7A, Figure 7B, Figure 7C or Table 1, or (ii) the order of promoter, enhancer and transgene elements can be as shown in Figure 1, Figure 7a, Figure 7b, Figure 7c or Table 1, or (iii) the sequence of the promoter, transgene, WPRE and poly(A) element is as shown in Figure 1, Figure 7a, Figure 7b, Figure 7c or may be as shown in Table 1, and optionally may be in the same order as shown in Figure 1, Figure 7a, Figure 7b, Figure 7c or Table 1, with or without enhancer elements, or (iv) promoter, transgene, WPRE, poly (A), the sequence of CMV intron (e.g. CMVint) elements can be as shown in Figure 1, Figure 7A, Figure 7B, Figure 7C or Table 1, optionally with or without enhancer elements in Figure 1, Figure 7A, Figure 7B , may be the same order as shown in Figure 7c or Table 1. In some embodiments, the orientation of any one of the elements is as shown in any one of the expression cassettes shown in Figures 1, 7A, 7B, 7C, and Table 1. The sequences of individual expression cassette elements discussed herein (e.g., promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, and chimeric introns) may include at least, for example, any one of the sequences of those elements provided herein. For example, it may be any of the sequences having 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the expression cassette includes one promoter described herein (with or without one or more enhancers described herein) that drives one copy of a transgene (e.g., any one of the transgenes described herein). Includes. In some embodiments, the expression cassette is one described herein, without any enhancer (e.g., without an enhancer described herein, e.g., without αMHce and/or ACTC1e) that leads to one copy of the transgene. Optionally, such expression cassette comprises an intronic element, such as a CMV intronic element, and/or a chimeric intron (e.g., Chim int described herein) and/or a codon optimized polynucleotide sequence. Includes a transgene with . In some embodiments, the expression cassette is one described herein, without any enhancer (e.g., without an enhancer described herein, e.g., without αMHce and/or ACTC1e) that leads to one copy of the transgene. and optionally further comprises a CMV intron element, and/or a chimeric intron (e.g., Chim int). In some embodiments, the expression cassette is one described herein, without any enhancer (e.g., without an enhancer described herein, e.g., without αMHce and/or ACTC1e) that leads to one copy of the transgene. A promoter, and optionally, further includes a CMV intron. In some embodiments, the expression cassette is one described herein, without any enhancer (e.g., without an enhancer described herein, e.g., without αMHce and/or ACTC1e) that leads to one copy of the transgene. A promoter, wherein the transgene comprises a codon-optimized polynucleotide sequence. In some embodiments, the expression cassette comprises one promoter described herein and one, two or more enhancers described herein that drive expression of one copy of the transgene (e.g., αMHCe and /or contains the ACTC1e enhancer). In some embodiments, the expression cassette includes one promoter described herein and one enhancer described herein (e.g., an αMHce or ACTC1e enhancer) that drives expression of one copy of the transgene. In some embodiments, the expression cassette comprises one promoter described herein and two enhancers described herein operably linked to one copy of the transgene (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e). In some embodiments, the promoter is a cardiac-specific promoter, such as the human cTnT promoter (such as the short human promoter, hcTnTp) and/or the chicken cTnT promoter (such as ccTnTp). In some embodiments where one or more enhancers are used, the enhancer is a cardiac-specific enhancer, such as αMHce and/or ACTC1e. In some embodiments, two or more cardiac-specific enhancers are used, where two or more of the enhancers may be the same or different (e.g., both or both αMHce, both or both ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardio-specific enhancers are used, where the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e) . In some embodiments, the expression cassette includes at least two enhancers in the order of first αMHCe and then ACTC1e. In some embodiments, the expression cassette includes at least two enhancers in the order of first ACTC1e and then αMHCe. In some embodiments, the transgene comprises a non-codon optimized polynucleotide sequence encoding the gene product. In some embodiments, the transgene comprises a codon optimized polynucleotide sequence encoding the gene product. In some embodiments, one or more intronic elements are also used in addition to promoter and enhancer elements. In some embodiments, CMV intron elements are used. In some implementations, chimeric intron elements (Chim int) are used. In some embodiments, both CMV introns and chimeric introns (Chim int) are used. In some embodiments where one promoter is used, the order of elements is as shown in any of the expression cassettes shown in Figure 7B. For example, (i) the order of the promoter and transgene elements can be as shown in Figure 7B, (ii) the order of the promoter, enhancer and transgene elements can be as shown in Figure 7B, or (iii) the promoter , the order of the transgene, WPRE and poly(A) elements may be as shown in Figure 7b, optionally with or without enhancer elements, or (iv) the promoter, transgene, WPRE The order of the , poly(A), CMV intron (e.g. CMVint) elements may be as shown in Figure 7B, and optionally may be in the same order as shown in Figure 7B, with or without enhancer elements. In some embodiments where one promoter is used, the orientation of either element is as shown in either of the expression cassettes shown in Figure 7B. In some implementations, the orientation of the elements is a forward orientation. In some implementations, the orientation of the elements is reverse orientation. The sequences of individual expression cassette elements discussed herein (e.g., promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, and chimeric introns) may include at least, for example, any one of the sequences of those elements provided herein. For example, it may be any of the sequences having 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the expression cassette is an expression cassette described herein (with or without one or more enhancers described herein) that drives expression of two copies of a transgene (e.g., any one of the transgenes described herein). Contains a promoter. In some embodiments, the expression cassette comprises two promoters described herein (with or without one or more enhancers described herein), each promoter linked to a transgene (e.g., one of the transgenes described herein). is operably linked to one copy of In some embodiments, the expression cassette is described herein without any enhancer (e.g., without any enhancer described herein, e.g., without αMHce and/or ACTC1e) that directs expression of two copies of the transgene. Contains the two promoters described in . In some embodiments, the expression cassette comprises two promoters described herein and one, two or more enhancers described herein that drive expression of two copies of the transgene (e.g., αMHCe and /or contains the ACTC1e enhancer). In some embodiments, the expression cassette comprises two promoters described herein and two enhancers described herein operably linked to two copies of the transgene (e.g., comprising an αMHCe and/or ACTC1e enhancer) ), where each transgene is operably linked to one promoter and one enhancer. In some embodiments, the promoter is a cardiac-specific promoter, such as the human cTnT promoter (such as the short human promoter, hcTnTp) and/or the chicken cTnT promoter (such as ccTnTp). In embodiments where two promoters are used, the two promoters may be the same or different. In some embodiments in which two promoters drive the expression of two copies of the transgene (each promoter driving the expression of one copy of the transgene), both promoters may be the same cardiac-specific promoter or may be different from each other. You can. In some embodiments, both promoters can be the human cTnT promoter (e.g., both can be the short human promoter, hcTnTp). In some embodiments, both promoters can be chicken cTnT promoter (eg, ccTnT). In some embodiments, the two promoters are different, for example, one is a human cTnT promoter (e.g., human cTnT short promoter, hcTnTp) and one is a chicken cTnT promoter (e.g., ccTnT). In some embodiments where two promoters and two transgenes are used, the two transgenes may be the same or different (eg, may be the same or different variants of the same transgene). For example, the first copy of the transgene can be a non-codon optimized polynucleotide sequence encoding the gene product and the second copy of the transgene can be a codon optimized polynucleotide sequence encoding the gene product. In some embodiments, the two copies of the transgene used in the expression cassette are identical. In some embodiments where one or more enhancers are used, the enhancer is a cardiac-specific enhancer, such as αMHce and/or ACTC1e. In some embodiments, two or more cardiac-specific enhancers are used, where two or more of the enhancers may be the same or different (e.g., both or both αMHce, both or both ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardio-specific enhancers are used, where the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e) . In some embodiments where two promoters are used, two cardiac-specific enhancers operably linked to the transgene are also used, optionally where one enhancer is αMHce and the other enhancer is ACTC1e. In some embodiments, the expression cassette includes at least two enhancers in the order of first αMHCe and then ACTC1e. In some embodiments, the expression cassette includes at least two enhancers in the order of first ACTC1e and then αMHCe. In some embodiments where two promoters are used, one or more intronic elements are also used. In some embodiments where two promoters are used, CMV intronic elements are also used. In some embodiments where two promoters are used, chimeric intronic elements (Chim int) are also used. In some embodiments where two promoters are used, a CMV intron and a chimeric intron (Chim int) are used. In some embodiments where two promoters are used, the order of the elements is as shown in either of the expression cassettes shown in Figure 7C. For example, (i) the order of the promoter and transgene elements can be as shown in Figure 7C, (ii) the order of the promoter, enhancer and transgene elements can be as shown in Figure 7C, or (iii) the promoter , the order of the transgene, WPRE and poly(A) elements may be as shown in Figure 7C, optionally with or without enhancer elements, or (iv) the promoter, transgene, WPRE , the order of the poly(A), CMV intron (e.g. CMVint) and chimeric intron (e.g. Chim int) elements may be as shown in Figure 7C, optionally with or without enhancer elements, and may be in the same order as shown in Figure 7C. . In some embodiments where two promoters are used, the first promoter and associated transgene (and optionally enhancer) are oriented in a forward 5' to 3' direction, and the second promoter and associated transgene (and optionally enhancer) are oriented in a forward 5' to 3' direction. It is oriented in the reverse direction. In some embodiments where two promoters are used, the first promoter and associated transgene (and optionally enhancer) are oriented in reverse with respect to the 5' to 3' direction, and the second promoter and associated transgene (and optionally enhancer) are oriented in reverse with respect to the 5' to 3' direction. ) is oriented in the forward direction. In some embodiments where two promoters are used, both the promoter and the associated transgene (and, optionally, the enhancer) are oriented in the forward 5' to 3' direction. In some embodiments where two promoters are used, both the promoter and the associated transgene (and, optionally, the enhancer) are oriented in reverse with respect to the 5' to 3' direction. In some embodiments where two promoters are used, the orientation of either element (e.g., forward or reverse in the 5' to 3' direction) is as shown in either of the expression cassettes shown in Figure 7C. In some embodiments where two promoters are used, the orientation of any one of the promoter, if any, enhancer, transgene, WPRE, poly(A), CMV intron and chimeric intron elements (e.g., forward orientation in the 5' to 3' direction) or reverse) as shown in either of the expression cassettes shown in Figure 7C. The sequences of individual expression cassette elements discussed herein (e.g., promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, and chimeric introns) may include at least, for example, any one of the sequences of those elements provided herein. For example, it may be any of the sequences having 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity.

Expression cassette sequences of the present disclosure can be found, without limitation, in Table 1. In some embodiments, the expression cassette comprises about 3.2 kilobases (kb), 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb or less. In some embodiments, the expression cassette is about 1.9 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.0 kb, 3.1 kb, 3.2 kb, or Includes more.

In some embodiments, the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20-24 and SEQ ID NOs: 45-63. It contains polynucleotide sequences that share a. In some embodiments, the expression cassette is a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:64-75 Includes. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:61. do. In some embodiments, the expression cassette includes SEQ ID NO:61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:62. do. In some embodiments, the expression cassette includes SEQ ID NO:62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:63. do. In some embodiments, the expression cassette includes SEQ ID NO:63. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 49. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 51. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:55. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 56. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 57. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 58. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 59. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:60. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:67. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:69. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. do. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:75. do. In some such embodiments, the sequence encoding DWORF (DWORF open reading frame) may be replaced with a sequence encoding another polypeptide described herein, and the sequence identity referenced above may be replaced by a polynucleotide sequence encoding DWORF (DWORF open reading frame). It does not take into account the part of the decoding frame.

In some embodiments, the transgene within the expression cassette encodes a polypeptide for use in treating or preventing a heart disease or disorder. In some embodiments, the transgene within the expression cassette is: DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulator 3 (BAG3), alpha-crystallin B chain (CRYAB), LMNA (e.g., lamin A and lamin C isoforms), troponin I type 3 (TNNI3), phospholamban (PLN), lysosome-associated membrane protein 2 (LAMP2, such as LAMP2a, LAMP2b and LAMP2c isoforms), desmoplakin (DSP, such as DPI and DPII isoforms), desmoglein 2 (DSG2), and junctional plakoglobin (JUP), or variants of any one of these polypeptides (e.g., at least 75%, at least 85%, at least 95% thereof) %, having a sequence identity of at least 97% or at least 99%). In some embodiments, the transgene within the expression cassette encodes DWORF (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes JPH2 (e.g., full-length JPH2 or an N-terminal fragment of JPH2) (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes BAG3 (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes CRYAB (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes an LMNA lamin A isoform (or variant thereof). In some embodiments, the transgene within the expression cassette encodes an LMNA lamin C isoform (or variant thereof). In some embodiments, the transgene within the expression cassette encodes TNNI3 (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes PLN (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes LAMP2a (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes LAMP2b (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes LAMP2c (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes a DSP DPI isoform (or variant thereof). In some embodiments, the transgene within the expression cassette encodes a DSP DPII isoform (or variant thereof). In some embodiments, the transgene within the expression cassette encodes DSG2 (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes JUP (or a variant thereof). In some embodiments, the transgene within the expression cassette encodes a human polypeptide (e.g., one of the human polypeptides described herein).

In some embodiments, the expression cassettes described herein result in cardiac cell specific expression of the transgene. In some embodiments, the expression cassettes described herein result in cardiomyocyte-specific expression of the transgene. In some embodiments, the expression cassettes described herein can be used to achieve high expression of the transgene in heart cells (e.g., cardiomyocytes) and low or no expression in other cells (e.g., low expression in liver cells). or no expression, low expression or no expression in muscle cells other than cardiac muscle cells, low expression or no expression in cardiac fibroblasts). In some embodiments, the expression cassettes described herein enable high expression of a transgene in cardiac tissue of a subject (e.g., in a human heart). In some embodiments, the expression cassettes described herein allow no or low expression of the transgene in tissues of the subject other than the heart (e.g., liver or muscle other than tissue of the heart). “High” and “low” may be relative to each other, e.g., expression of the transgene in cardiac cells (e.g., cardiomyocytes) and/or cardiac tissue may be related to expression of the transgene in other cells and tissues (e.g., , liver, and muscles excluding the heart) may be at least 2-, 5-, 10-, 15-, 20-, 50-, 100-, 150-, or 200-fold higher.

vector

In some aspects, the present disclosure provides vectors comprising the expression cassettes provided herein. The vector can be any viral vector or any non-viral vector known in the art or described herein.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV), an adenoviral vector (AV), a lentiviral vector (LV), a retroviral vector (RV), a herpes simplex virus vector (HSV), or a poxvirus vector. am.

In some embodiments, provided herein is an AAV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an AV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an LV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an RV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an HSV comprising any of the expression cassettes described herein. In some embodiments, provided herein are poxvirus-based vectors comprising any of the expression cassettes described herein.

In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a lipid vector comprising liposomal or plasmid DNA and a lipid solution.

For example, viral and non-viral vectors and delivery systems are described in Sung & Kim 2019, Biomaterials Research 23:8; Mali, 2013, Indian Journal of Human Genetics, 19(1):3-8; Hardee et al., 2017, Genes 8:65; Bulcha et al., 2020, Signal Transduction and Targeted Therapy; Ghosh et al., 2020, Applied Biosafety: Journal of ABSA International 25(1):7-18, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, the vector is a recombinant vector.

In some embodiments, the vectors described herein comprise an expression cassette comprising a polynucleotide encoding any one of the gene products described herein. In some embodiments, the expression cassette comprises SEQ ID NO: 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225 and at least 75%, 80%, 90%, 95%, 96 %, 97%, 98%, 99%, or 100% identity.

In some embodiments, the vectors described herein include an expression cassette comprising a polynucleotide encoding DWORF. In some embodiments, the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20-24 and SEQ ID NOs: 45-75. It contains polynucleotide sequences that share a.

In some aspects of the disclosure, vectors are used to deliver expression cassettes described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy. In some embodiments, the present disclosure provides a polynucleotide encoding a gene product (e.g., any one of the gene products described herein, e.g., a DWORF polypeptide) operably linked to a promoter and a pharmaceutically acceptable carrier. It provides a viral vector containing an expression cassette containing. In some embodiments, the present disclosure provides a polynucleotide encoding a gene product (e.g., any one of the gene products described herein, e.g., a DWORF polypeptide) operably linked to a capsid and a promoter and a pharmaceutically acceptable A virion comprising an expression cassette containing a possible carrier is provided. In some embodiments, the present disclosure provides a polynucleotide encoding a gene product (e.g., any one of the gene products described herein, e.g., a DWORF polypeptide) operably linked to a promoter and a pharmaceutically acceptable carrier. It provides a plasmid containing an expression cassette containing a.

In some embodiments, the viral vectors described herein are replication defective in that they are unable to further replicate and package their genomes independently. For example, when cardiac cells are targeted with a virion, the transgene is expressed in the targeted cardiac cells, but due to the fact that the targeted cardiac cells lack packaging and accessory function genes, the virions are unable to replicate. . In some embodiments, the viral vectors described herein are replication competent.

In some embodiments, vectors described herein can be delivered to both dividing and non-dividing cells. In some embodiments, vectors described herein can be delivered to non-dividing cells. In some embodiments, vectors described herein can be delivered to dividing cells.

In some embodiments, a vector comprising an expression cassette described herein results in cardiac cell specific expression of a transgene. In some embodiments, a vector comprising an expression cassette described herein results in cardiomyocyte-specific expression of a transgene. In some embodiments, vectors comprising the expression cassettes described herein can be used to achieve high expression of the transgene in heart cells (e.g., cardiomyocytes) and low or no expression in other cells (e.g., liver cells). low expression or no expression in, low expression or no expression in muscle cells other than cardiac muscle cells, low expression or no expression in cardiac fibroblasts). In some embodiments, vectors comprising expression cassettes described herein enable high expression of a transgene in cardiac tissue of a subject (e.g., in a human heart). In some embodiments, vectors comprising the expression cassettes described herein allow no or low expression of the transgene in tissues of the subject other than the heart (e.g., liver or muscle other than tissue of the heart). . “High” and “low” may be relative to each other, e.g., expression of the transgene in cardiac cells (e.g., cardiomyocytes) and/or cardiac tissue may be related to expression of the transgene in other cells and tissues (e.g., , liver, and muscles excluding the heart) may be at least 2-, 5-, 10-, 15-, 20-, 50-, 100-, 150-, or 200-fold higher than the expression in the liver.

Recombinant AAV virions

In some aspects, the present disclosure provides recombinant AAV (rAAV) virions comprising the expression cassettes provided herein. In some embodiments, the rAAV virion includes a capsid protein and an expression cassette. In some embodiments, the expression cassette comprises a polynucleotide encoding any of the gene products described herein.

In some embodiments, the expression cassette includes a polynucleotide encoding DWORF. In some embodiments, the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20-24 and SEQ ID NOs: 45-63. It contains polynucleotide sequences that share a. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:61. do. In some embodiments, the expression cassette includes SEQ ID NO:61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:62. do. In some embodiments, the expression cassette includes SEQ ID NO:62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:63. do. In some embodiments, the expression cassette includes SEQ ID NO:63.

In some aspects of the disclosure, rAAV virions are used to deliver the expression cassettes described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy. Accordingly, the present disclosure provides a rAAV virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operably linked to a promoter and a pharmaceutically acceptable carrier.

The rAAV virion of the present disclosure includes a capsid protein. The capsid protein is a structural protein that makes up the assembled icosahydral package of the rAAV virion containing the expression cassette. Capsid proteins are classified according to serotype. The wild-type capsid serotype in the rAAV virion can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al., BioDrugs 31:317 -334 (2017)). Engineered capsid types include chimeric capsids and mosaic capsids (Choi et al., Curr Gene Ther. 5: 299-310 (2005)). Capsids are selected for rAAV virions based on their ability to transduce specific tissues or cell types (Liu et al., Curr Pharm Des. 21:3248-56 (2015)).

As described herein, any capsid protein that can facilitate rAAV virion transduction into cardiac cells can be used for delivery of the transgene. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells resulting in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al., Mol. Ther. 16 :P1073-1080 (2008)). Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery into cardiac cells resulting in high expression (see US 63/012,703, the contents of which are incorporated herein by reference). Other capsid proteins with various characteristics may also be used in the rAAV virions of the present disclosure. AAV vectors and capsids are described in US Patent Publication No. US10011640B2; US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2, US10480011B2, and US10894949B2, the contents of which are incorporated herein by reference; and International Patent Publication Nos. WO2020198737A1, WO2019028306A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1, WO2017212019A1, No. WO2020117898A1, No. WO2017192750A1, No. WO2020191300A1, and No. WO2017100671A1 ( Their contents are incorporated herein by reference.

In some embodiments, rAAV virions of the present disclosure comprise engineered capsid proteins. The engineered capsid protein can be derived from a parent, e.g., a wild-type capsid, and comprise a variant polypeptide sequence relative to the parent capsid sequence, e.g., at one or more sites. For example, variant regions of the parent capsid may occur in the VR-IV region, VR-V region, VR-VII region, and/or VR-VIII region (e.g., Buening and Srivastava. Mol Ther Methods Clin Dev . 12:248-265 (2019)).

In some embodiments, the capsid protein is an AAV5/AAV9 chimeric capsid protein. In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide fragments derived from the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide fragments derived from the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, at least one polypeptide segment is derived from AAV5 capsid protein and at least one polypeptide segment is derived from AAV9 capsid protein.

In some embodiments, the capsid protein is a combined capsid protein. As used herein, “combination capsid protein” refers to the AAV5/AAV9 chimeric capsid protein, which further comprises amino acid variations relative to the chimeric parent sequence at one or more sites. In some embodiments, one or more regions of the chimeric parent sequence are selected from regions equivalent to the VR-IV region, VR-V region, VR-VII region, and VR-VIII region of the AAV9 capsid protein.

In some embodiments, the rAAV virion comprises an engineered capsid protein selected from Table 7.

In some embodiments, rAAV is replication defective, in that the rAAV virion is unable to further replicate and package its genome independently. For example, when cardiac cells are targeted with rAAV virions, the transgene is expressed in the targeted cardiac cells, but due to the fact that the targeted cardiac cells lack the AAV rep and cap genes and accessory function genes, rAAV It cannot be copied.

In some embodiments, rAAV virions of the present disclosure encapsulating an expression cassette as described herein can be produced using helper-free production. rAAV is a replication-deficient virus and generally requires the components of a live helper virus, such as adenovirus, in host cells for packaging of infectious rAAV virions. The rAAV helper-free production system allows the production of infectious rAAV virions without the use of live helper viruses. In the helper-free system, the host packaging cell line is co-transfected with three plasmids. The first plasmid may contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) required for packaging of rAAV virions. The second plasmid may contain the necessary AAV genes (eg, REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs. The host packaging cell line may be, for example, an AAV-293 host cell. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmid. In some embodiments, the CAP gene may encode an AAV capsid protein, e.g., as described herein.

In some embodiments, the CAP gene may encode an AAV capsid protein, e.g., as described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a cTnT promoter sequence. In some embodiments, the polypeptide of interest is a DWORF polypeptide.

Expression cassettes, enhancers and/or promoters described herein in the context of AAV virions need not be limited to their use in AAV virions, and may be used in any manner that essentially requires expression of a polynucleotide encoding a gene product. It may be incorporated into other constructs.

rAAV virions can deliver a transgene to a subject's cells, which are eventually expressed in the cells. Transgenes delivered by rAAV virions can be incorporated into the genome of targeted cells, allowing potential long-term expression of the transgene product. Compared to other viral transgene delivery systems such as adenovirus, rAAV virions have the advantage of low immunogenicity. rAAV virions can be used to transduce and deliver transgenes to many cell types, including eye, blood, liver, heart, joint tissue, muscle, brain kidney, or lung cells (U.S. Pat. No. 10,308,957; U.S. Pat. No. 9,803,218). rAAV virions can contain a genome of up to about 5.2 kilobases (kb), which limits the size of polynucleotides that can be incorporated into host cells to about 4.4 kb (Choi et al., Mol Brain . 7:1 ( 2014)).

How to use

How to Increase Polypeptide Expression

The present disclosure provides a method of increasing polypeptide expression in a cell, the method comprising contacting the cell with either a vector or a virion (e.g., a rAAV virion) described herein. In some embodiments, the cells are cardiac cells. In some embodiments, the cells are cardiomyocytes. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the polypeptide is any polypeptide for use in treating or preventing heart disease. In some embodiments, the polypeptide is any of the polypeptides described herein. In some embodiments, the polypeptide is encoded by any one of the transgenes described herein.

The present disclosure provides a method of increasing polypeptide expression in a tissue, the method comprising contacting the tissue with either a vector or a virion (e.g., a rAAV virion) described herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

The present disclosure provides a method of increasing polypeptide expression in an organ, the method comprising contacting the organ with any of the vectors or virions (e.g., rAAV virions) described herein. In some embodiments, the organ is the heart. In some embodiments, the heart has disease or is at risk for disease. In some embodiments, the heart has borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the heart contains a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable polypeptide expression compared to a healthy heart. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

The present disclosure provides a method of increasing polypeptide expression in a subject, comprising administering to the subject either a vector or a virion (e.g., rAAV virion) described herein. In some embodiments, the subject is an animal. The animal may be, but is not limited to, a mouse, rat, dog, or non-human primate. In some embodiments, the subject is a human. In some embodiments, polypeptide expression is increased in the heart of the subject. In some embodiments, the subject has heart disease or is at risk for heart disease. In some embodiments, the subject has borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to a healthy subject.

In some embodiments, the polypeptide is expressed in a cell, tissue, organ, or subject at a desired level of expression. The “desired expression level” may be selected such that the expression level is comparable to the expression of the polypeptide in a healthy or diseased cell, tissue, organ or subject. For example, an increased level of polypeptide expression compared to diseased cardiac cells, cardiac tissue, heart, or a subject with a cardiac disease or disorder may be the desired level of expression. The desired expression level can be expressed as the difference in expression achieved between different vectors or virions (e.g., rAAV virions) containing different expression cassettes. For example, a vector or virion containing an expression cassette containing a promoter and an enhancer (e.g., rAAV virion) may be compared to a vector or virion containing only a promoter (e.g., rAAV virion). level of expression can be achieved.

The level of polypeptide expression achieved by any vector or virion (e.g., rAAV virion) comprising an expression cassette can be described as a “fold” change (i.e., increase or decrease) compared to basal polypeptide expression. . The level of polypeptide expression achieved by a vector or virion (e.g., rAAV virion) comprising an expression cassette can be compared to the polypeptide expression achieved by an expression cassette comprising a single promoter, no enhancer, and a sequence encoding the polypeptide. Comparatively, it can be described as a “fold change”. Fold change is a relative amount such that the expression level between the expression level achieved by the expression cassette and the reference expression level is expressed as a ratio. When describing a fold change in polypeptide expression, “about” is understood to refer to ±0.5-fold. Polypeptide expression levels can be classified as “low expression,” “medium expression,” or “high expression.” “Low expression” is meant to include expression levels between about 1.5-fold and 20-fold increase in polypeptide expression. “Medium expression” is meant to include expression levels between about a 20-fold increase and about a 60-fold increase in polypeptide expression. “High expression” is meant to include expression levels between about 60-fold and 140-fold increase in polypeptide expression.

In some embodiments, the level of polypeptide expression is increased by about 1.5-fold to 150-fold. In some embodiments, the level of polypeptide expression is at least about 1.5-fold, about 3.5-fold, about 5.5-fold, about 7.5-fold, about 9.5-fold, about 11.5-fold, about 13.5-fold, about 15.5-fold, about 17.5-fold, about 19.5-fold, About 21.5 times, about 23.5 times, about 25.5 times, about 27.5 times, about 29.5 times, about 31.5 times, about 33.5 times, about 35.5 times, about 37.5 times, about 39.5 times, about 41.5 times, about 43.5 times, about 45.5 times times, about 47.5 times, about 49.5 times, about 51.5 times, about 53.5 times, about 55.5 times, about 57.5 times, about 59.5 times, about 61.5 times, about 63.5 times, about 65.5 times, about 67.5 times, about 69.5 times, About 71.5 times, about 73.5 times, about 75.5 times, about 77.5 times, about 79.5 times, about 81.5 times, about 83.5 times, about 85.5 times, about 87.5 times, about 89.5 times, about 91.5 times, about 93.5 times, about 95.5 times times, about 97.5 times, about 99.5 times, about 101.5 times, about 103.5 times, about 105.5 times, about 107.5 times, about 109.5 times, about 111.5 times, about 113.5 times, about 115.5 times, about 117.5 times, about 119.5 times, About 121.5 times, about 123.5 times, about 125.5 times, about 127.5 times, about 129.5 times, about 131.5 times, about 133.5 times, about 135.5 times, about 137.5 times, about 139.5 times, about 141.5 times, about 143.5 times, about 145.5 times, about 147.5 times, or about 149.5 times.

In some embodiments, the level of polypeptide expression is increased by at least about 5-fold. In some embodiments, the level of polypeptide expression is increased by at least about 10-fold. In some embodiments, the level of polypeptide expression is increased by at least about 15-fold. In some embodiments, the level of polypeptide expression is increased by at least about 25-fold. In some embodiments, the level of polypeptide expression is increased by at least about 35-fold. In some embodiments, the level of polypeptide expression is increased by at least about 50-fold. In some embodiments, the level of polypeptide expression is increased by at least about 60-fold. In some embodiments, the level of polypeptide expression is increased by at least about 75-fold. In some embodiments, the level of polypeptide expression is increased by at least about 85-fold. In some embodiments, the level of polypeptide expression is increased by at least about 100-fold. In some embodiments, the level of polypeptide expression is increased by at least about 110-fold. In some embodiments, the level of polypeptide expression is increased by at least about 125-fold.

In some embodiments, the fold increase is for an expression cassette comprising a single promoter, no enhancer, and a sequence encoding a polypeptide. In some embodiments, the fold increase is relative to healthy cells, tissues, organs, or subjects. In some embodiments, the fold increase is relative to a cell, tissue, organ, or subject having a disease.

How to Increase DWORF Expression

The present disclosure provides a method of increasing DWORF expression in a cell, the method comprising contacting the cell with a rAAV virion described herein. In some embodiments, the cells are cardiac cells. In some embodiments, the cells are cardiomyocytes. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

The present disclosure provides a method of increasing DWORF expression in a cell, the method comprising contacting a tissue with a rAAV virion described herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

The present disclosure provides a method of increasing DWORF expression in an organ, the method comprising contacting the organ with a rAAV virion described herein. In some embodiments, the organ is the heart. In some embodiments, the heart has disease or is at risk of disease. In some embodiments, the heart has borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the heart contains a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable DWORF expression compared to a healthy heart. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting occurs in vivo.

The present disclosure provides a method of increasing DWORF expression in a subject, the method comprising administering to the subject a rAAV virion described herein. In some embodiments, the subject is an animal. The animal may be, but is not limited to, a mouse, rat, dog, or non-human primate. In some embodiments, the subject is a human. In some embodiments, DWORF expression is increased in the heart of the subject. In some embodiments, the subject has heart disease or is at risk for heart disease. In some embodiments, the subject has borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to a healthy subject.

In some embodiments, DWORF is expressed in a cell, tissue, organ, or subject at a desired level of expression. The “desired expression level” may be selected such that the expression level is comparable to DWORF expression in healthy or diseased cells, tissues, organs or subjects. For example, an increased level of DWORF expression compared to diseased cardiac cells, cardiac tissue, heart, or a subject with a cardiac disease or disorder may be the desired level of expression. The desired expression level can be expressed as the expression difference achieved between different rAAV virions containing different expression cassettes. For example, rAAV virions containing an expression cassette containing a promoter and an enhancer can achieve a desired level of expression compared to rAAV virions containing only a promoter.

The level of DWORF expression achieved by a rAAV virion containing the expression cassette can be described as a “fold” change (i.e., increase or decrease) compared to basal DWORF expression. The level of DWORF expression achieved by, for example, a rAAV virion containing an expression cassette is a "fold change" compared to the DWORF expression achieved by an expression cassette containing a single promoter, no enhancer, and a sequence encoding DWORF. It can be described as. Fold change is a relative amount such that the expression level between the expression level achieved by the expression cassette and the reference expression level is expressed as a ratio. When describing a fold change in DWORF expression, “about” is understood to refer to ±0.5 fold. DWORF expression levels can be classified as “low expression,” “medium expression,” or “high expression.” “Low expression” is meant to include expression levels between about 1.5-fold and 20-fold increase in DWORF expression. “Medium expression” is meant to include expression levels between about a 20-fold increase and about a 60-fold increase in DWORF expression. “High expression” is meant to include expression levels between about 60-fold and 140-fold increase in DWORF expression.

In some embodiments, DWORF expression levels are increased by about 1.5-fold to 150-fold. In some embodiments, the DWORF expression level is about 1.5-fold, about 3.5-fold, about 5.5-fold, about 7.5-fold, about 9.5-fold, about 11.5-fold, about 13.5-fold, about 15.5-fold, about 17.5-fold, about 19.5-fold, about 21.5 times, about 23.5 times, about 25.5 times, about 27.5 times, about 29.5 times, about 31.5 times, about 33.5 times, about 35.5 times, about 37.5 times, about 39.5 times, about 41.5 times, about 43.5 times, about 45.5 times , about 47.5 times, about 49.5 times, about 51.5 times, about 53.5 times, about 55.5 times, about 57.5 times, about 59.5 times, about 61.5 times, about 63.5 times, about 65.5 times, about 67.5 times, about 69.5 times, about 71.5 times, about 73.5 times, about 75.5 times, about 77.5 times, about 79.5 times, about 81.5 times, about 83.5 times, about 85.5 times, about 87.5 times, about 89.5 times, about 91.5 times, about 93.5 times, about 95.5 times , about 97.5 times, about 99.5 times, about 101.5 times, about 103.5 times, about 105.5 times, about 107.5 times, about 109.5 times, about 111.5 times, about 113.5 times, about 115.5 times, about 117.5 times, about 119.5 times, about 121.5 times, about 123.5 times, about 125.5 times, about 127.5 times, about 129.5 times, about 131.5 times, about 133.5 times, about 135.5 times, about 137.5 times, about 139.5 times, about 141.5 times, about 143.5 times, about 145.5 times , increases by about 147.5 times, or about 149.5 times.

In some embodiments, the level of DWORF expression is increased by at least about 5-fold. In some embodiments, the level of DWORF expression is increased by at least about 10-fold. In some embodiments, the level of DWORF expression is increased by at least about 15-fold. In some embodiments, the level of DWORF expression is increased by at least about 25-fold. In some embodiments, the level of DWORF expression is increased by at least about 35-fold. In some embodiments, the level of DWORF expression is increased by at least about 50-fold. In some embodiments, DWORF expression levels are increased by at least about 60-fold. In some embodiments, the level of DWORF expression is increased by at least about 75-fold. In some embodiments, the level of DWORF expression is increased by at least about 85-fold. In some embodiments, the level of DWORF expression is increased by at least about 100-fold. In some embodiments, DWORF expression levels are increased by at least about 110-fold. In some embodiments, the level of DWORF expression is increased by at least about 125-fold.

In some embodiments, the fold increase is for an expression cassette containing a single promoter, no enhancer, and a sequence encoding DWORF. In some embodiments, the fold increase is relative to healthy cells, tissues, organs, or subjects. In some embodiments, the fold increase is relative to a cell, tissue, organ, or subject having a disease.

Treatment method

In one aspect, any of the vectors containing the expression cassettes described herein can be used to treat a disease, such as heart disease.

In one aspect, rAAV virions containing the expression cassettes described herein can be used to treat diseases (Wang et al . Nat Rev Drug Discov. 18:358-378 (2019)). For treatment, rAAV virions contain microdystrophin (Chamberlain et al . Mol Ther. 25:1125-1131 (2017)), glial cell line-derived neurotrophic factor (McFarthing et al . J Parkinsons Dis. 9:251-264 (2019)), and It has been used to deliver transgenes encoding polypeptides such as Factor IX (Nathwani et al. N Engl J Med. 371:1994-2004 (2014)).

Various strategies to treat heart failure using rAAV-based delivery of transgenes have been explored in vivo. In a porcine model of heart failure, the contractile regulator β-adrenergic receptor was targeted by delivery of βARKct, a small polypeptide that indirectly prevents disruption of β-adrenergic receptor signaling (Raake et al. Eur Heart J. 34:1437-47 ( 2013)). In a canine model, cardiomyocyte viability was improved by rAAV-based delivery of vascular endothelial growth factor (VEGF) isoforms. In human clinical trials, rAAV-based delivery to the heart of SERCA2a, an isoform of the SERCA calcium pump, has been tested as a treatment for heart failure. SERCA, or Sarco/endoplasmic reticulum Ca ²⁺ -ATPase, or SR Ca ²⁺ -ATPase, is a calcium ATPase-type P-ATPase. SERCA is present in the sarcoplasmic reticulum (SR) within muscle cells. Ca ²⁺ ATPase transfers Ca ²⁺ from the cell's cytosol to the lumen of the SR at the expense of ATP hydrolysis during muscle relaxation. SERCA activity is required for proper contractile function of the heart. However, direct replacement of SERCA activity by rAAV-based delivery of SERCA2a isoforms did not show significant effects in clinical trials (Bass-Stringer et al., Heart, Lung and Circulation . 27:1285-1300 (2018)). Enhancing SERCA activity using alternative strategies is required for the treatment of cardiac diseases such as heart failure and cardiomyopathy.

The cytoplasmic face of SERCA has three major domains: a phosphorylation and nucleotide binding domain that forms the catalytic site, and an actuator domain that is involved in the transmission of major conformational changes. The rate at which SERCA moves Ca ²⁺ across the SR membrane can be regulated by the regulatory protein phospholamban (PLN). SERCA is generally inhibited by and is closely related to PLN. Increased β-adrenergic stimulation reduces the association between SERCA and PLN by phosphorylation of PLN by PKA. When PLN is associated with SERCA, the rate of Ca ²⁺ transport is reduced; Upon dissociation of PLN, Ca ²⁺ transport increases.

An alternative strategy to enhance SERCA activity by delivering SERCA2a isoforms is to enhance the activity of naturally expressed SERCA by displacing PLN. Contacting SERCA with the aforementioned DWORF polypeptide can displace PLN and enhance SERCA activity.

In some embodiments, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising an expression cassette comprising a polynucleotide encoding a therapeutic polypeptide operably linked to a promoter. and administering an effective amount of a vector, wherein the therapeutic polypeptide can be any polypeptide useful for treating a heart disease. As described herein, the vector can be any viral or non-viral vector.

In some embodiments, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding DWORF operably linked to a promoter. It includes administering an effective amount of rAAV virion, which is a recombinant adeno-associated virus (rAAV) virion comprising.

In methods of treating a subject as described herein, “treatment” or “treatment of a condition in a subject in need of treatment” refers to: (1) beneficial or desirable results, including clinical results such as symptom reduction; taking steps to obtain; (2) Inhibition of the disease, e.g., stopping or reducing the occurrence of the disease or its clinical symptoms; (3) alleviating the disease, e.g., causing regression of the disease or its clinical symptoms; or (4) delayed disease. For the purposes of the methods described herein, beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms associated with heart failure, cardiomyopathy, dilated cardiomyopathy, myocardial infarction, acute myocardial infarction, and chronic myocardial infarction. .

In another aspect, the disclosure provides a method of preventing a cardiac disease or disorder in a subject in need thereof, the method comprising an expression cassette comprising a polynucleotide encoding a therapeutic polypeptide operably linked to a promoter. and administering an effective amount of a vector, wherein the therapeutic polypeptide can be any polypeptide useful for preventing heart disease. As described herein, the vector can be any viral or non-viral vector. In some embodiments, preventing the disease prevents clinical symptoms of the disease from developing in a patient who is at risk for the disease but does not yet experience or exhibit symptoms of the disease.

Subjects in need of treatment using the compositions and methods of the present invention include, but are not limited to, subjects suffering from or at risk of heart failure. A subject “suffering from” heart failure is considered to have symptoms or confirmed diagnosis associated with any of the heart diseases described herein. A subject “at risk” is considered to have one or more risk factors associated with any of the heart diseases described herein.

In some embodiments, the methods described herein are useful for treating heart disease or disorders with reduced ejection fraction (HFrEF). In some embodiments, the methods described herein are useful for treating heart disease or disorders with preserved ejection fraction (HFpEF).

In some embodiments, the methods described herein are useful for treating cardiomyopathy. In some embodiments, the methods described herein are useful for treating dilated cardiomyopathy. In some embodiments, the subject suffers from or is at risk for cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy (DCM). In some embodiments, the DCM is genetic DCM (e.g., DCM associated with a PLN mutation in the subject to be treated). In some embodiments, the methods described herein are useful for treating PLN mutation-related cardiomyopathy. In some embodiments, the DCM is non-hereditary DCM. In some embodiments, the subject is suffering from or at risk of myocardial infarction. In some embodiments, the myocardial infarction is chronic myocardial infarction. In some embodiments, the myocardial infarction is an acute myocardial infarction.

The cardiomyopathy phenotype may develop in a subject through a number of molecular mechanisms. Transgenic animals have been developed to investigate the molecular pathophysiology of specific mechanisms and the efficacy of potential therapeutic and preventive strategies against cardiomyopathy phenotypes (Law et al., J Clin Med. 9:520 (2020)). These animal models can be used to evaluate aspects of the rAAV viral genome, rAAV virion, and compositions thereof described herein. The MLP ^-/- transgenic mouse model reproduces the phenotype of dilated cardiomyopathy, for example, by knocking out muscle LIM protein, a positive regulator of myogenic differentiation associated with the actin-based cytoskeleton. The absence of LIM proteins results in disruption of cytoskeletal structure and reduced Ca ²⁺ cycling (Minamisawa et al . Cell. 99:313-22 (1999)). Artificial replacement with a phosphomimetic PLN transgene delivered by rAAV reduced DCM symptoms in animals, including improved ejection fraction (Iwanaga Y et al . J Clin Invest. 113:727-736 (2004)). Although the MLP ^-/- model recapitulates the DCM phenotype in a general manner, other transgenic mouse models of DCM are more suited to specific DCM phenotypes, such as those driven by mutations to the PLN gene. For example, a transgenic mouse model was developed to reproduce the clinically observed PLN-R14Del mutation (Haghighi et al., Proc. Natl. Acad. Sci. USA 103:1388-1393 (2006)). Mouse models with different transgenic modifications to induce a DCM phenotype are not interchangeable in assessing the efficacy of a given therapeutic or preventive strategy, and each model is expected to provide different information for translation of that therapy. .

In some embodiments, the subject in need of treatment has a genetic risk allele (i.e., mutation) for a heart disease or disorder. The risk allele may be, for example, a mutation in the PLN gene. Mutations in the PLN gene can cause dysfunctional inhibitory effects on SERCA activity. Clinically observable mutations in the PLN gene and protein include mutations in the PLN promoter, PLN ^L39stop mutations, abnormal R9C, R9L, and R9H mutations, PLN gene duplications, and deletion of arginine 14 (R14del) in the regulatory domain of PLN. Includes cutting. Each of these mutations was directly associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, or arrhythmic right ventricular cardiomyopathy (Table 8). (Landstrom et al., Am Heart J. 161:165-171 (2011), Lee et al., Cardiol Young 24 :953-954 (2014); Haghighi et al., J. Clin. Invest. 111:869-876 (2003); Schmitt et al., Science 299:1410-1413 (2003); Haghighi et al., Proc. Natl. Acad. Sci. USA 103:1388-1393 (2006); Medeiros A et al., Am. Heart J. 162:1088-1095 (2011)).

Various mutations in PLN have different mechanisms leading to cardiomyopathy phenotypes. For example, the R9C mutation indirectly blocks phosphorylation of PLN by PKA and prevents the formation of monomeric PLN that can bind to SERCA. In some embodiments, the subject in need of treatment has a PLN promoter mutation. In some embodiments, the subject in need of treatment has a PLN ^L39stop mutation. In some embodiments, the subject in need of treatment has an R9C mutation. In some embodiments, the subject in need of treatment has an R9L mutation. In some embodiments, the subject in need of treatment has an R9H mutation. In some embodiments, the subject in need of treatment has a PLN gene copy. In some embodiments, the subject in need of treatment has an R14del mutation.

Mutations can be detected by many types of genetic analysis known in the art. Genetic analysis may be performed, for example, by direct sequencing, fluorescence in situ hybridization assay, polymerase chain reaction-based assay, nucleotide microarray assay, or any other method known in the art to determine the sequence characteristics of polynucleotides sampled from a subject. It could be technology. For example, DNA was isolated from peripheral blood samples of patients diagnosed with dilated cardiomyopathy (DCM) or arrhythmic right ventricular cardiomyopathy (ARVC). The coding region of the PLN gene among the isolated DNA was sequenced using the BigDye Terminator DNA sequencing kit (version 2.0) on a 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). All patients diagnosed with DCM or ARVC had a PLN R14del mutation (van der Zwaag et al. Eur J Heart Fail. 14:1199-1207 (2012)).

How to Reduce, Improve and Prevent Symptoms

In some embodiments, the disclosure provides a method of reducing one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering any of the vectors comprising an expression cassette described herein. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administering the subject a vector comprising an expression cassette described herein. In some embodiments, the present disclosure provides a method of reducing one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering a rAAV virion described herein. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of rAAV virions to the subject. In some embodiments, the heart disease or disorder is heart failure. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the heart disease or disorder is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the heart disease or disorder is chronic myocardial infarction. In some embodiments, the heart disease or disorder is acute myocardial infarction.

In some embodiments, the present disclosure provides a method of improving one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering a vector comprising an expression cassette described herein. In some embodiments, the symptoms are improved compared to the symptoms of the heart disease or disorder prior to administration of the vector to the subject. In some embodiments, the present disclosure provides a method of ameliorating one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering a rAAV virion described herein. In some embodiments, the symptoms are improved compared to the symptoms of the heart disease or disorder prior to administration of rAAV virions to the subject. In some embodiments, the heart disease or disorder is heart failure. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the heart disease or disorder is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the heart disease or disorder is chronic myocardial infarction. In some embodiments, the heart disease or disorder is acute myocardial infarction.

In some embodiments, the disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering a vector comprising an expression cassette described herein. In some embodiments, the present disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject, the method comprising administering a rAAV virion described herein. In some embodiments, the condition is prevented in a subject considered to be at risk for a heart disease or disorder. In some embodiments, the heart disease or disorder is heart failure. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the heart disease or disorder is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the heart disease or disorder is chronic myocardial infarction. In some embodiments, the heart disease or disorder is acute myocardial infarction.

In some embodiments, the symptoms are reduced compared to the symptoms of the heart disease or disorder prior to administering the vector to the subject. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of rAAV virions to the subject. In some embodiments, the heart disease or disorder is heart failure. In some embodiments, the heart disease or disorder is cardiomyopathy. In some embodiments, the heart disease or disorder is dilated cardiomyopathy. In some embodiments, the heart disease or disorder is myocardial infarction. In some embodiments, the heart disease or disorder is chronic myocardial infarction. In some embodiments, the heart disease or disorder is acute myocardial infarction.

As used herein, “symptoms” include, for example, any of the diagnostic criteria or symptoms associated with heart disease described herein. The severity and changes in symptoms and diagnostic findings are determined by a qualified health care professional who provides an evaluation and analyzes the results of such evaluation.

Common symptoms in subjects with or at risk for heart disease are fatigue, shortness of breath, edema, chest pain, arrhythmias, blood clots, impaired heart valve function, and heart murmurs. In some embodiments, the subject experiences reduced symptoms associated with the cardiac disease described herein following administration of a vector, rAAV virion, or composition of the present disclosure. In some embodiments, the improved symptom is one or more of the following: improved contractility; Reduces fatigue; Reduced shortness of breath; Reduces edema; Reduces chest pain; Reduces arrhythmias; Reduces blood clots; Improved heart valve function; Reduces heart noise. In some embodiments, the symptom is a change in 6-minute walking distance. In some embodiments, symptoms are determined by the Minnesota Living with Heart Failure Questionnaire. In some embodiments, the condition is abnormal levels of B-type natriuretic peptide ( i.e., BNP, NT-proBNP). In some embodiments, the severity of symptoms is determined by measuring LV remodeling. In some embodiments of the methods described herein, it improves one or more measures of cardiac function. In some embodiments, measurements of cardiac function include fractional shortening and/or left ventricular internal dimension (LVID). In some embodiments, the measurement of cardiac function includes left ventricular end-systolic volume (LVESV). In some embodiments, the improvement in cardiac function is ejection fraction. In some embodiments, improvement in cardiac function is observed between weeks 2 and 12. In some embodiments, the method reduces cardiac remodeling. In some embodiments, the method responds to reducing DWORF expression in a subject suffering from myocardial infarction.

Ejection fraction is a measure of the percentage of blood that leaves the heart each time it contracts. Ejection fraction is determined using stroke volume (SV) and end-diastolic volume (EDV), and is calculated as EF(%) = (SV/EDV)Х100. Ejection fraction can be measured in a subject by imaging tests, including echocardiogram, cardiac catheterization, magnetic resonance imaging (MRI), computed tomography (CT), and/or nuclear medicine scan. Normal ejection fraction is about 50% to about 75%. “Borderline” ejection fraction may range from about 41% to about 50%. The reduced ejection fraction is less than about 41%. Borderline or reduced ejection fraction can be used as a symptom to diagnose a heart disease or disorder. It will be appreciated that the cutoff values between normal, borderline, and reduced ejection fraction are approximate and will be ultimately determined by a person skilled in the art, e.g., a cardiologist.

In some embodiments of the methods provided herein, it may be desirable to improve ejection fraction. If the ejection fraction percentage increases, the ejection fraction may be considered improved. In some embodiments, ejection fraction

In some embodiments of the methods provided herein, it may be desirable to preserve ejection fraction. Preserving ejection fraction prevents the development of a heart disease or disorder, prevents the progression of a heart disease or disorder, prevents worsening of symptoms associated with a heart disease or disorder, or prevents heart disease or disorders in subjects at risk for heart disease or disorder. It can be used to prevent worsening of symptoms associated with a disease or disorder.

The present disclosure provides methods for improving ejection fraction in subjects suffering from or at risk for cardiac disease or disorders. In some embodiments, the ejection fraction is improved (i.e., increased) in a subject following administration of a vector comprising an expression cassette described herein. In some embodiments, the ejection fraction is improved (i.e., increased) in a subject following administration of a vector or rAAV virion described herein. In some embodiments, the ejection fraction is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks after administration of the vector or rAAV virion to the subject. week, at approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 22 weeks, or approximately 24 weeks. It improves. In some embodiments, the ejection fraction is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% after administration of the vector or rAAV virion to the subject. %, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% It improves.

The present disclosure provides a method of preserving ejection fraction in a subject suffering from or at risk of cardiac disease or disorder. For example, a subject may maintain an ejection fraction that would be expected to decrease without administration of a vector or rAAV virion or pharmaceutical composition of the present disclosure. In some embodiments, ejection fraction is preserved in the subject following administration of a vector or rAAV virion of the present disclosure. In some embodiments, the ejection fraction is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks after administration of the vector or rAAV virion to the subject. week, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 22 weeks or about 24 weeks. preserved. In some embodiments, the ejection fraction is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% after administration of the vector or rAAV virion to the subject. %, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% It is preserved as much as

Assessment of cardiac contractility can be used to evaluate acute and chronic forms of heart failure. Cardiac contractility can be monitored by invasive hemodynamic monitoring, continuous ECG monitoring, central venous pressure, renal function, pulse oximetry, arterial pressure monitoring, pulmonary artery catheterization, and/or transesophageal echocardiography (Kuhn C, Werdan K. Surgical Treatment: Evidence-Based and Problem-Oriented. Munich: Zuckschwerdt; 2001. Available from https://www.ncbi.nlm.nih.gov/books/NBK6895/).

Dyspnea and fatigue associated with the heart conditions described herein can be measured using questionnaires. The Modified Pulmonary Functional Status and Dyspnea Questionnaire (PFSDQ-M)10 (Huang et al. Am J Crit Care. 17:436-442 (2008)) and the Minnesota Living in Heart Failure Questionnaire (Minnesota Life in Heart Failure Patients). The Living with Heart Failure Questionnaire (MLHFQ) 11 (Bilbao et al., Health Qual Life Outcomes. 14:23 (2016)) can be used, for example, to measure subjects with heart disease as described herein. . The questionnaire is self-administered and allows for a score that is used to assess symptom severity of shortness of breath, fatigue, and other heart health-related symptoms.

Cardiomyopathy, myocardial infarction, and heart valve function can be assessed by exercise stress testing, electrocardiogram, echocardiogram, chest X-ray, cardiac CT scan, or angiogram with cardiac catheterization, cardiac MRI, and blood B-type natriuretic peptide ( BNP) levels, and/or genetic screening. Additional tests are needed to diagnose certain types of cardiomyopathy, myocardial infarction, or heart valve dysfunction.

In some embodiments, administering a vector comprising an expression cassette described herein to a subject improves the subject's athletic performance (e.g., improves running distance and/or time to exhaustion). In some embodiments, administering to a subject a rAAV comprising an expression cassette described herein encoding DWORF improves the subject's exercise capacity (e.g., improving running distance and/or time to exhaustion).

Dilated cardiomyopathy (DCM) is a progressive disease of the heart muscle characterized by chamber hypertrophy and contractile dysfunction of the left ventricle in the absence of chronic pressure and/or volume overload. DCM is primarily diagnosed using echocardiography.

Echocardiography using PLAV view in 2D/M-mode is used to measure several parameters including ejection fraction, LVIDd/s, IVSd, LVPWd, and fractional shortening. These parameters are used to evaluate left ventricular cavity size, wall thickness, and radial function. Diagnostic criteria for DCM include LVIDd/s greater than 112% (2 SD) corrected for age and body surface area (BSA). Fractional shortening of less than 25% is the criterion for diagnosing DCM in the presence of dilated ventricles (Mathew et al. Echo Res Pract. 4:G1-G13 (2017)).

Qualitative assessment of left and right ventricular structure and function with special reference to radial and longitudinal function and regional wall motion abnormalities is assessed by echocardiography in the apical four-chamber (A4C) view in 2D mode. Ejection fraction (EF) can be estimated using, for example, the two-sided Simpsons method. An EF of less than 45% is the diagnostic criterion for DCM in the presence of dilated ventricles (Mathew et al . Echo Res Pract. 4: G1-G13 (2017)).

administration

In some embodiments, the vectors and compositions of the present disclosure can be administered to a subject in need thereof by systemic application, for example, by intravenous, intraarterial, or intraperitoneal delivery. In some embodiments, rAAV virions and compositions of the present disclosure can be administered to subjects in need thereof, for example, by intravenous, intraarterial, or intraperitoneal delivery of vectors similar to those shown in animal models (Katz et al., Gene Ther 19:659-669 (2012)). In some embodiments, the vectors, rAAV virions, and compositions of the present disclosure treat or prevent heart failure. In some embodiments of cardiomyopathy, the viral vector is administered systemically. In some embodiments, rAAV virions are administered by intravenous or intraarterial injection.

The present disclosure provides methods for expressing polypeptides in cells in vitro, ex vivo, or in vivo. In some embodiments, the present disclosure provides methods for expressing DWORF polypeptides in cells in vitro, ex vivo, or in vivo. Methods include exposing target cells to vectors, rAAV virions, or pharmaceutical compositions, e.g., described herein. Target cells may be, for example, without limitation, cardiac cells, muscle cells, induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), and/or cardiomyocytes. In some embodiments, a method of expressing a polypeptide (e.g., a DWORF polypeptide) in a cell includes transfecting or transducing a target cell or target cell population with a vector described herein (referred to as “transfecting”). ) includes. In some embodiments, a method of expressing a polypeptide (e.g., a DWORF polypeptide) in a cell includes transducing a target cell or target cell population with a rAAV virion or pharmaceutical composition described herein (“infecting” (referred to as ). In some embodiments, rAAV transduces cardiac cells. In some embodiments, rAAV transduces cardiomyocytes. In some embodiments, rAAV transduces induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs).

In some embodiments, vector transfection or transduction increases polypeptide expression in the heart of the subject. In some embodiments, rAAV transduction increases DWORF polypeptide expression in the heart of the subject. “Increased polypeptide expression” generally refers to expression of at least 5%, 10%, 15%, 20% or more compared to a control subject or tissue not treated with the vector. “Increased DWORF polypeptide expression” generally refers to expression of at least 5%, 10%, 15%, 20% or more compared to a control subject or tissue not treated with the vector. In some embodiments, detectable expression means expression that is 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater compared to a non-vector control. Expression can be assessed by Western blot, or enzyme-linked immunosorbent assay (ELISA), as described in the examples below, or other methods known in the art. In some cases, expression is measured quantitatively using standard curves. Standard curves can be generated using purified proteins, such as purified DWORF polypeptides, by methods described in the Examples or known in the art. Alternatively, expression of the therapeutic gene product can be assessed by quantification of the corresponding mRNA. In some embodiments, the method results in expression of a polypeptide (e.g., a DWORF polypeptide) in the heart of the subject.

In some embodiments, the method does not result in detectable expression of the polypeptide in the subject's heart, liver, and/or muscles other than cardiac fibroblasts. In some embodiments, the method results in expression of the polypeptide in cardiomyocytes.

In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in muscles of the subject other than the heart. In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in the liver of the subject. In some embodiments, the method results in expression of a DWORF polypeptide in cardiomyocytes. In some embodiments, the method does not result in detectable expression of the DWORF polypeptide in cardiac fibroblasts.

In some embodiments, the increased polypeptide expression in cardiac tissue is 3 Х 10 ¹⁴ vg/kg or less, 2 Х 10 14 vg/kg or less, 1 Х ^{10 14 vg} /kg or less, 9 per kg body weight of the subject. Х 10 ¹³ vg/kg or less, 8 Х 10 ¹³ vg/kg or less, 7 Х 10 ¹³ vg/kg or less, 6 Х 10 ¹³ vg/kg or less, 5 Х 10 ¹³ vg/kg or less, 4 Х 10 ¹³ vg/ kg or less, 3 Х 10 ¹³ vg/kg or less, 2 Х 10 ¹³ vg/kg or less, or 1 Х 10 ¹³ vg/kg or less in units of vector genome (vg).

In some embodiments, the increased DWORF polypeptide expression in cardiac tissue is less than or equal to 3 Х 10 ¹⁴ vg/kg, less than or equal to 2 Х 10 ¹⁴ vg/kg, or less than or equal to 1 Х 10 ¹⁴ vg/kg per kg of body weight of the subject. 9 Х 10 ¹³ vg/kg or less, 8 Х 10 ¹³ vg/kg or less, 7 Х 10 ¹³ vg/kg or less, 6 Х 10 ¹³ vg/kg or less, 5 Х 10 ¹³ vg/kg or less, 4 Х 10 ¹³ vg or less It occurs at a dose of less than or equal to Х 10 ¹³ vg/kg, less than or equal to 2 Х 10 ¹³ vg/kg, or less than or equal to 1 Х 10 ¹³ vg/kg in units of vector genome (vg).

Pharmaceutical compositions and kits

Vectors of the present disclosure are generally delivered to a subject as a pharmaceutical composition. In some embodiments, rAAV virions of the present disclosure are delivered to a subject as a pharmaceutical composition. Pharmaceutical compositions include a pharmaceutically acceptable solvent (eg, water, etc.) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer at approximately neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS at a pH of about 7). The pharmaceutical composition may include a pharmaceutically acceptable salt. The concentration of salt may be selected to ensure that the pharmaceutical composition is isotonic to the target tissue or nearly isotonic to the target tissue.

In various embodiments, the compositions described herein contain pharmaceutically acceptable vehicles (e.g., carriers, diluents, and excipients) for injectable formulations. These may be particularly isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts), or dry, especially lyophilized compositions, which, when added, are dissolved in sterilized water. Or, in the case of physiological saline, it allows the composition of an injectable solution. Exemplary pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions; Formulations containing sesame oil, peanut oil, or aqueous propylene glycol; Sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

In various embodiments, the pharmaceutical compositions of the present disclosure have a molecular weight of about 1 Х 10 ⁸ genome copies per milliliter (GC/mL), about 5 Х 10 ⁸ GC/mL, about 1 Х 10 ⁹ GC/mL, about 5 Х 10 ⁹ GC/mL, approximately 1 Х 10 ¹⁰ GC/mL, approximately 5 Х 10 ¹⁰ GC/mL, approximately 1 Х 10 ¹¹ GC/mL, approximately 5 Х 10 ¹¹ GC/mL, approximately 1 Х 10 ¹² GC/mL, approximately 5 Х 10 ¹² GC/mL, about 5 Х 10 ¹³ GC/mL, about 1 Х 10 ¹⁴ GC/mL, or about 5 Х 10 ¹⁴ GC/mL of viral vector (e.g., rAAV virion).

In various embodiments, the pharmaceutical compositions of the present disclosure contain about 1 Х 10 ⁸ viral genome per milliliter (vg/mL), about 5 Х 10 ⁸ vg/mL, about 1 Х 10 ⁹ vg/mL, about 5 Х 10 ⁹ vg/mL, approximately 1 Х 10 ¹⁰ vg/mL, approximately 5 Х 10 ¹⁰ vg/mL, approximately 1 Х 10 ¹¹ vg/mL, approximately 5 Х 10 ¹¹ vg/mL, approximately 1 Х 10 ¹² vg/mL, approximately 5 Х 10 ¹² vg/mL, about 5 Х 10 ¹³ vg/mL, about 1 Х 10 ¹⁴ vg/mL, or about 5 Х 10 ¹⁴ vg/mL of a viral vector (e.g., rAAV virion). .

In some embodiments, the pharmaceutical compositions of the present disclosure have a volume of about 1 mL, 5 mL, 10 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, About 55 mL, about 60 mL, 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL , about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about It is administered in a total volume of 180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about 210 mL, about 215 mL, or about 220 mL.

Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR), using a standard curve generated with reference samples with known concentrations of the polynucleotide genome of the virus. For AAV, the reference sample used is often the transfer plasmid used for production of rAAV virions, but other reference samples may be used.

Alternatively or additionally, the concentration of viral vector can be determined by measuring the titer of the vector on a cell line. Virus titer is generally expressed as virus particles per unit volume (vp) (e.g., vp/mL). In various embodiments, the pharmaceutical compositions of the present disclosure produce about 1 Х 10 ⁸ viral particles per milliliter (vp/mL), about 5 Х 10 ⁸ vp/mL, about 1 Х 10 ⁹ vp/mL, about 5 Х 10 ⁹ vp/mL, approximately 1 Х 10 ¹⁰ vp/mL, approximately 5 Х 10 ¹⁰ vp/mL, approximately 1 Х 10 ¹¹ vp/mL, approximately 5 Х 10 ¹¹ vp/mL, approximately 1 Х 10 ¹² vp/mL, approximately 5 Х 10 ¹² vp/mL, about 5 Х 10 ¹³ vp/mL, about 1 Х 10 ¹⁴ vp/mL, or about 5 Х 10 ¹⁴ vp/mL of viral vector (e.g., rAAV virion). .

In some embodiments, the present disclosure provides a kit comprising a container containing a pharmaceutical composition as described herein.

Numbered Embodiments of the Invention (I)

Embodiment 1: A recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising a capsid protein and an expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter. A recombinant adeno-associated virus (rAAV) virion comprising, wherein the expression cassette is flanked by inverted terminal repeats.

Embodiment 2: The method of Embodiment 1, wherein the DWORF polypeptide is at least 90%, 95%, 96%, 97%, 98%, or rAAV virions, sharing 99% identity.

Embodiment 3: The rAAV virion of Embodiment 1, wherein the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

Embodiment 4: The rAAV virion of Embodiment 1 or 2, wherein the promoter is the chicken cTnT promoter.

Embodiment 5: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

Embodiment 6: The rAAV virion of Embodiment 4, wherein the chicken cTnT promoter comprises SEQ ID NO: 11.

Embodiment 7: The rAAV virion of embodiment 1 or 2, wherein the promoter is the human cTnT promoter.

Embodiment 8: The rAAV virion of embodiment 7, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13 .

Embodiment 9: The rAAV virion of Embodiment 7, wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 10: The rAAV virion according to any one of embodiments 1 to 9, wherein the expression cassette further comprises one or more enhancers.

Embodiment 11: The rAAV virion of embodiment 10, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

Embodiment 12: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

Embodiment 13: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78.

Embodiment 14: The rAAV virion of embodiment 11, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

Embodiment 15: The rAAV virion of embodiment 11, wherein the αMHC enhancer comprises SEQ ID NO:79.

Embodiment 16: The rAAV virion of any of Embodiments 1-15, wherein the expression cassette further comprises an intron.

Embodiment 17: The rAAV virion of embodiment 16, wherein the intron is selected from a CMV intron and a chimeric intron.

Embodiment 18: The rAAV virion of embodiment 17, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

Embodiment 19: The rAAV virion of embodiment 17, wherein the CMV intron sequence comprises SEQ ID NO:80.

Embodiment 20: The rAAV virion of embodiment 17, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

Embodiment 21: The rAAV virion of embodiment 17, wherein the chimeric intron sequence comprises SEQ ID NO:81.

Embodiment 22: The rAAV virion of any of embodiments 1-21, wherein the expression cassette further comprises a WPRE sequence.

Embodiment 23: The rAAV virion of embodiment 22, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

Embodiment 24: The rAAV virion of embodiment 22, wherein the WPRE sequence comprises SEQ ID NO:26.

Embodiment 25: The rAAV virion of any of embodiments 1-24, wherein the expression cassette further comprises a polyadenylation sequence.

Embodiment 26: The rAAV virion of Embodiment 25, wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence.

Embodiment 27: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

Embodiment 28: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

Embodiment 29: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

Embodiment 30: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

Embodiment 31: The rAAV virion of any of embodiments 1-30, wherein the expression cassette is flanked by ITRs.

Embodiment 32: The rAAV virion of Embodiment 31, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15 on.

Embodiment 33: The rAAV virion of embodiment 31, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

Embodiment 34: The rAAV virion of any of embodiments 1-33, wherein the expression cassette comprises a single promoter.

Embodiment 35: The rAAV virion of any of embodiments 1-33, wherein the expression cassette comprises two promoters.

Embodiment 36: The rAAV virion of any of embodiments 1-35, wherein the expression cassette comprises a single copy of a sequence encoding a DWORF polypeptide.

Embodiment 37: The rAAV virion of any of embodiments 1-35, wherein the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide.

Embodiment 38: The rAAV virion of any of Embodiments 1-37, wherein the expression cassette comprises 1, 2, 3, or 4 enhancers.

Embodiment 39: The rAAV virion of any of embodiments 1 to 38, wherein the expression cassette comprises one or two introns.

Embodiment 40: The rAAV virion of any of embodiments 1 to 39, wherein the expression cassette comprises one or two WPRE sequences.

Embodiment 41: The rAAV virion of any of embodiments 1-40, wherein the expression cassette comprises one or two polyadenylation sequences.

Embodiment 42: The rAAV virion of any of embodiments 1-41, wherein the expression cassette comprises about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb or less.

Embodiment 43: The method of any one of embodiments 1 to 41, wherein the expression cassette is about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, A rAAV virion comprising about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

Embodiment 44: The method of embodiment 1, wherein the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97%, 98% with any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. A rAAV virion comprising polynucleotide sequences that share 99%, or 100% identity.

Embodiment 45: The rAAV virion of embodiment 1, wherein the expression cassette comprises any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75.

Embodiment 46: The method of embodiment 1, wherein the expression cassette is a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61 Containing rAAV virions.

Embodiment 47: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:61.

Embodiment 48: The method of embodiment 1, wherein the expression cassette is a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:62 Containing rAAV virions.

Embodiment 49: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:62.

Embodiment 50: The method of embodiment 1, wherein the expression cassette is a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:63 Containing rAAV virions.

Embodiment 51: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:63.

Embodiment 52: The rAAV virion of any of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143).

Embodiment 53: The rAAV virion of any of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144).

Embodiment 54: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is a chimeric capsid protein.

Embodiment 55: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is an AAV5/AAV9 chimeric capsid protein.

Embodiment 56: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is selected from any of SEQ ID NOs: 145-200.

Embodiment 57: An expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter.

Embodiment 58: The expression cassette of embodiment 57, wherein the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

Embodiment 59: The expression cassette of embodiment 57 or 58, wherein the promoter is the chicken cTnT promoter.

Embodiment 60: The expression cassette of embodiment 59, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

Embodiment 61: The expression cassette of embodiment 59, wherein the chicken cTnT promoter comprises SEQ ID NO: 11.

Embodiment 62: The expression cassette of embodiment 57 or 58, wherein the promoter is the human cTnT promoter.

Embodiment 63: The expression cassette of embodiment 62, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 64: The expression cassette of embodiment 62, wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 65: The expression cassette of any one of embodiments 57-64, wherein the expression cassette further comprises one or more enhancers.

Embodiment 66: The expression cassette of embodiment 65, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

Embodiment 67: The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

Embodiment 68: The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78.

Embodiment 69: The expression cassette of embodiment 66, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

Embodiment 70: The expression cassette of embodiment 66, wherein the αMHC enhancer comprises SEQ ID NO:79.

Embodiment 71: The expression cassette of any of embodiments 57-70, wherein the expression cassette further comprises an intron.

Embodiment 72: The expression cassette of any of embodiments 57-70, wherein the intron is selected from a CMV intron and a chimeric intron.

Embodiment 73: The expression cassette of embodiment 72, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

Embodiment 74: The expression cassette of embodiment 72, wherein the CMV intron sequence comprises SEQ ID NO:80.

Embodiment 75: The expression cassette of embodiment 72, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

Embodiment 76: The expression cassette of embodiment 72, wherein the chimeric intron sequence comprises SEQ ID NO:81.

Embodiment 77: The expression cassette of any one of embodiments 57-76, wherein the expression cassette further comprises a WPRE sequence.

Embodiment 78: The expression cassette of embodiment 77, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

Embodiment 79: The expression cassette of embodiment 77, wherein the WPRE sequence comprises SEQ ID NO:26.

Embodiment 80: The expression cassette of any of embodiments 57-79, wherein the expression cassette further comprises a polyadenylation sequence.

Embodiment 81: The expression cassette of embodiment 80, wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence.

Embodiment 82: The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

Embodiment 83: The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence comprises SEQ ID NO: 27.

Embodiment 84: The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

Embodiment 85: The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence comprises SEQ ID NO: 28.

Embodiment 86: The expression cassette of any of embodiments 57-85, wherein the expression cassette is flanked by ITRs.

Embodiment 87: The expression cassette of embodiment 86, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15 .

Embodiment 88: The expression cassette of embodiment 86, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

Embodiment 89: The expression cassette of embodiments 57-88, comprising a single promoter.

Embodiment 90: The expression cassette of embodiments 57-88, comprising two promoters.

Embodiment 91: The expression cassette of any one of embodiments 57-90, comprising a single copy of a sequence encoding a DWORF polypeptide.

Embodiment 92: The expression cassette of any of embodiments 57-90, comprising two copies of a sequence encoding a DWORF polypeptide.

Embodiment 93: The expression cassette of any of embodiments 57-92, comprising 1, 2, 3, or 4 enhancers.

Embodiment 94: The expression cassette of any of embodiments 57-93, comprising 1 or 2 introns.

Embodiment 95: The expression cassette of any of embodiments 57-94, comprising one or two WPRE sequences.

Embodiment 96: The expression cassette of any one of embodiments 57-95, comprising one or two polyadenylation sequences.

Embodiment 97: The expression cassette of any of embodiments 57-96, comprising up to about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb.

Embodiment 98: The method of any one of embodiments 57-96, wherein the , an expression cassette comprising about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

Embodiment 99: The method of embodiment 1, wherein any one of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75 and at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or an expression cassette comprising polynucleotide sequences that share 100% identity.

Embodiment 100: The expression cassette of embodiment 57, comprising any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75.

Embodiment 101: The method of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61 , expression cassette.

Embodiment 102: The expression cassette of embodiment 57, comprising SEQ ID NO:61.

Embodiment 103: The method of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:62 , expression cassette.

Embodiment 104: The expression cassette of embodiment 57, comprising SEQ ID NO:62.

Embodiment 105: The method of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:63 , expression cassette.

Embodiment 106: The expression cassette of embodiment 57, comprising SEQ ID NO:63.

Embodiment 107: The expression cassette of any one of embodiments 57-98, further comprising a 5' inverted terminal repeat and a 3' inverted terminal repeat.

Embodiment 108: A pharmaceutical composition comprising the rAAV virion of any one of Embodiments 1 to 56 and a pharmaceutically acceptable diluent.

Embodiment 109: A kit comprising the pharmaceutical composition of embodiment 108.

Embodiment 110: A method of increasing DWORF expression in a cell, comprising contacting the cell with the rAAV virion of any of Embodiments 1 to 56 or the composition of Embodiment 108.

Embodiment 111: The method of embodiment 110, wherein the cells are cardiac cells.

Embodiment 112: The method of embodiment 111, wherein the cells are cardiomyocytes.

Embodiment 113: The method of any of embodiments 110-112, wherein DWORF expression is increased by about 1.5-fold to 150-fold.

Embodiment 114: The method of any of embodiments 110-113, wherein the contacting occurs in vitro.

Embodiment 115: The method of any of embodiments 110-113, wherein the contacting occurs in vivo.

Embodiment 116: A method of increasing DWORF expression in a tissue, comprising contacting the tissue with the rAAV virion of any of Embodiments 1-56 or the composition of Embodiment 108.

Embodiment 117: The method of embodiment 116, wherein the tissue is cardiac tissue.

Embodiment 118: The method of embodiment 116 or 117, wherein DWORF expression is increased by about 1.5-fold to 150-fold.

Embodiment 119: The method of any of embodiments 116-118, wherein the contacting occurs in vitro.

Embodiment 120: The method of embodiment 116 or 118, wherein the contacting occurs in vivo.

Embodiment 121: A method of increasing DWORF expression in an organ, comprising contacting the organ with the rAAV virion of any of Embodiments 1-56 or the composition of Embodiment 108.

Embodiment 122: The method of embodiment 121, wherein the organ is the heart.

Embodiment 123: The method of embodiment 122, wherein the individual has a heart condition or is at risk of developing the condition.

Embodiment 124: The method of embodiment 122 or 123, wherein the heart has a reduced or borderline ejection fraction.

Embodiment 125: The method of embodiment 122 or 123, wherein the heart has a normal ejection fraction.

Embodiment 126: The method of any one of embodiments 122-125, wherein the heart comprises a genetic mutation associated with heart disease.

Embodiment 127: The method of embodiment 126, wherein the gene mutation is a PLN mutation.

Embodiment 128: The method of any of embodiments 121-127, wherein the heart has low or undetectable levels of DWORF expression compared to a healthy heart.

Embodiment 129: The method of any of embodiments 121-128, wherein DWORF expression is increased by about 1.5-fold to 150-fold.

Embodiment 130: The method of any of embodiments 121-129, wherein the contacting occurs in vitro.

Embodiment 131: The method of any of embodiments 121-129, wherein the contacting occurs in vivo.

Embodiment 132: A method of increasing DWORF expression in a subject, comprising administering to the subject the rAAV virion of any of Embodiments 1-56 or the composition of Embodiment 108.

Embodiment 133: The method of embodiment 132, wherein the subject is an animal.

Embodiment 134: The method of embodiment 132, wherein the subject is a human.

Embodiment 135: The method of any one of embodiments 132-134, wherein DWORF expression is increased in the heart of the subject.

Embodiment 136: The method of any one of embodiments 132-135, wherein the subject has heart disease or is at risk for heart disease.

Embodiment 137: The method of any of embodiments 132-136, wherein the subject has borderline or reduced ejection fraction.

Embodiment 138: The method of any of embodiments 132-136, wherein the subject has a normal ejection fraction.

Embodiment 139: The method of any one of embodiments 132-138, wherein the subject has a genetic mutation associated with heart disease.

Embodiment 140: The method of embodiment 139, wherein the genetic mutation is a PLN mutation.

Embodiment 141: The method of any of embodiments 132-140, wherein the subject has low or undetectable levels of DWORF expression compared to a healthy subject.

Embodiment 142: A method of treating a cardiac disease or disorder in a subject in need thereof, comprising administering to the subject a rAAV virion of any one of embodiments 1 to 56 or a composition of embodiment 108. Method, including.

Embodiment 143: The method of embodiment 142, wherein the subject has a heart disease or disorder.

Embodiment 144: The method of embodiment 142, wherein the subject is at risk of developing a heart disease or disorder.

Embodiment 145: The method of any one of embodiments 142-144, wherein the heart disease or disorder is cardiomyopathy.

Embodiment 146: The method of any one of embodiments 142-144, wherein the heart disease or disorder is dilated cardiomyopathy.

Embodiment 147: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is myocardial infarction.

Embodiment 148: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is chronic myocardial infarction.

Embodiment 149: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is acute myocardial infarction.

Embodiment 150: The method of any one of embodiments 142-149, wherein the subject has a genetic risk allele for a heart disease or disorder.

Embodiment 151: The method of any one of embodiments 142-150, wherein the genetic risk allele comprises a mutation to the PLN gene.

Embodiment 152: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN promoter mutation.

Embodiment 153: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN ^L39stop mutation.

Embodiment 154: The method of embodiment 151, wherein the mutation to the PLN gene is an RC9 mutation.

Embodiment 155: The method of embodiment 151, wherein the mutation to the PLN gene is an R9L mutation.

Embodiment 156: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN gene duplication.

Embodiment 157: The method of embodiment 151, wherein the mutation to the PLN gene is an R14del mutation.

Embodiment 158: The method of any one of embodiments 142-157, wherein the cardiac disease or disorder is reduced ejection fraction (HFrEF).

Embodiment 159: The method of any of embodiments 142-157, wherein the cardiac disease or disorder has preserved ejection fraction (HFpEF).

Embodiment 160: The method of any one of embodiments 142-159, wherein the method causes expression of the DWORF polypeptide in the heart of the subject.

Embodiment 161: The method of any one of embodiments 142-160, wherein the method causes expression of the DWORF polypeptide in cardiomyocytes.

Embodiment 162: The method of any one of embodiments 142-161, wherein the method does not result in detectable expression of the DWORF polypeptide in muscles of the subject other than the heart.

Embodiment 163: The method of any one of embodiments 142-162, wherein the method does not result in detectable expression of the DWORF polypeptide in the liver of the subject.

Embodiment 164: The method of any one of embodiments 142-163, wherein the method does not result in detectable expression of the DWORF polypeptide in cardiac fibroblasts.

Embodiment 165: The method of any of embodiments 142-164, wherein the method improves one or more measures of cardiac function, optionally fractional shortening and/or left ventricular internal dimension (LVID).

Embodiment 166: The method of any one of embodiments 142-165, wherein an improvement in cardiac function is observed from week 2 to week 16.

Embodiment 167: The method of any one of embodiments 142-166, wherein the method reduces cardiac remodeling.

Embodiment 168: The method of any one of embodiments 142-166, wherein the method corresponds to a decrease in DWORF expression in a subject suffering from or at risk of heart disease.

Embodiment 169: The method of any one of embodiments 142-168, wherein the rAAV virion is administered by systemic administration.

Embodiment 170: The method of embodiment 169, wherein the systemic administration is selected from intravenous or intraluminal injection.

Embodiment 171: The method of embodiment 169 or 170, wherein the rAAV is administered as a unit dose.

Embodiment 172: The method of embodiment 171, wherein the unit dose is less than or equal to about 3Х10 ¹⁴ vg/kg, less than or equal to about 2Х10 ¹⁴ vg/kg, less than or equal to about 1Х10 ¹⁴ vg/kg, less than or equal to about 9Х10 ¹³ vg/kg, or less than or equal to about 8Х10 ¹³ vg. /kg or less, about 7Х10 ¹³ vg/kg or less, about 6Х10 ¹³ vg/kg or less, about 5Х10 ¹³ vg/kg or less, about 4Х10 ¹³ vg/kg or less, about 3Х10 ¹³ vg/kg or less, about 2Х10 ¹³ vg/kg or less or less, or comprising less than or equal to about 1Х10 ¹³ vg/kg.

Embodiment 173: A method of alleviating one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering the rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108. A method comprising steps.

Embodiment 174: A method of improving one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering the rAAV virion of any of Embodiments 1 to 56 or the composition of Embodiment 108. A method comprising steps.

Embodiment 175: A method of preventing one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering the rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108. A method comprising steps.

Embodiment 176: An expression cassette comprising a polynucleotide comprising a 5' to 3' arrangement of an element, wherein the element is:

i. one or more promoters;

ii. Optionally one or more enhancers;

iii. optionally one or more introns;

iv. One or more transgenes;

v. Optionally one or more WPRE sequences; and

vi. An expression cassette, optionally comprising one or more polyadenylation sequences, p(A).

Implementation 177: The method of implementation 176, wherein the 5' to 3' arrangement of the elements is:

vii. 5'-Promoter-Intron-Transgene-WPRE-p(A)-3';

viii. 5'-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

ix. 5'-Enhancer-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

x. 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

xi. 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

xii. 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';

xiii. 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';

xiv. 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

xiv. 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

xvi. 5'-Promoter-Intron-Transgene-WPRE-p(A)-Promoter-Intron-Transgene-p(A)-3'; and

xvii. An expression cassette selected from 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

Embodiment 178: The method of embodiment 176 or embodiment 177, wherein the transgene is a polynucleotide having an arrangement of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. An expression cassette having an increased expression level compared to a second expression cassette comprising.

Embodiment 179: The expression cassette of embodiment 178, wherein the level of increased expression is from about 1.5-fold to about 150-fold compared to the second expression cassette.

Embodiment 180: A recombinant adeno-associated virus (rAAV) virion, comprising a viral genome comprising a capsid protein and the expression cassette of any of embodiments 176 to 179, wherein the expression cassette is flanked by inverted terminal repeats. A recombinant adeno-associated virus (rAAV) virion.

Embodiment 181: The rAAV of embodiment 180, wherein the expression cassette comprises a transgene, wherein the transgene encodes a polypeptide for use in treating or preventing heart disease or alleviating symptoms associated with heart disease.

Embodiment 182: The rAAV of embodiment 180 or embodiment 181, wherein the capsid protein is selected from any one of SEQ ID NOs: 145-200.

Numbered Embodiments of the Invention (II)

Embodiment 1: A recombinant adeno-associated virus (rAAV) virion, comprising a viral genome comprising a capsid protein and an expression cassette comprising a polynucleotide sequence encoding a polypeptide operably linked to a promoter, wherein the expression The cassette is flanked by inverted terminal repeats, optionally wherein the polypeptide is a recombinant adeno-associated virus (rAAV) virion for expression in cardiac cells or tissues and/or for use in the treatment or prevention of cardiac disease. on.

Embodiment 2: The method of Embodiment 1, wherein the polypeptide is selected from the group consisting of DWORF, JPH2, BAG3, CRYAB, lamin A isoform of LMNA, lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, rAAV virions selected from the DPII isoforms of DSP, DSG2, and JUP.

Embodiment 3: The method of embodiment 1 or 2, wherein the polypeptide has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the polypeptide sequences in Tables 2a and 2b. Shared, rAAV virions.

Implementation Example 4: Implementation Example 1 The rAAV virion according to any one of to 3, wherein the promoter is the chicken cTnT promoter.

Embodiment 5: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

Embodiment 6: The rAAV virion of Embodiment 4, wherein the chicken cTnT promoter comprises SEQ ID NO: 11.

Implementation Example 7: Implementation Example 1 The rAAV virion according to any one of to 3, wherein the promoter is the human cTnT promoter.

Embodiment 8: The rAAV virion of embodiment 7, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13 .

Embodiment 9: The rAAV virion of Embodiment 7, wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 10: The rAAV virion according to any one of embodiments 1 to 9, wherein the expression cassette further comprises one or more enhancers.

Embodiment 11: The rAAV virion of embodiment 10, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

Embodiment 12: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

Embodiment 13: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78.

Embodiment 14: The rAAV virion of embodiment 11, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

Embodiment 15: The rAAV virion of embodiment 11, wherein the αMHC enhancer comprises SEQ ID NO:79.

Embodiment 16: The rAAV virion of any of Embodiments 1-15, wherein the expression cassette further comprises an intron.

Embodiment 17: The rAAV virion of embodiment 16, wherein the intron is selected from a CMV intron and a chimeric intron.

Embodiment 18: The rAAV virion of embodiment 17, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

Embodiment 19: The rAAV virion of embodiment 17, wherein the CMV intron sequence comprises SEQ ID NO:80.

Embodiment 20: The rAAV virion of Embodiment 17, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

Embodiment 21: The rAAV virion of embodiment 17, wherein the chimeric intron sequence comprises SEQ ID NO:81.

Embodiment 22: The rAAV virion of any of embodiments 1-21, wherein the expression cassette further comprises a WPRE sequence.

Embodiment 23: The rAAV virion of embodiment 22, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

Embodiment 24: The rAAV virion of embodiment 22, wherein the WPRE sequence comprises SEQ ID NO:26.

Embodiment 25: The rAAV virion of any of embodiments 1-24, wherein the expression cassette further comprises a polyadenylation sequence.

Embodiment 26: The rAAV virion of Embodiment 25, wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence.

Embodiment 27: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

Embodiment 28: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

Embodiment 29: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

Embodiment 30: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

Embodiment 31: The rAAV virion of any of embodiments 1-30, wherein the expression cassette is flanked by ITRs.

Embodiment 32: The rAAV virion of Embodiment 31, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15 on.

Embodiment 33: The rAAV virion of embodiment 31, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

Embodiment 34: The rAAV virion of any of embodiments 1-33, wherein the expression cassette comprises a single promoter.

Embodiment 35: The rAAV virion of any of embodiments 1-33, wherein the expression cassette comprises two promoters.

Embodiment 36: The rAAV virion of any of embodiments 1-35, wherein the expression cassette comprises a single copy of the sequence encoding the polypeptide.

Embodiment 37: The rAAV virion of any of embodiments 1-35, wherein the expression cassette comprises two copies of the sequence encoding the polypeptide.

Embodiment 38: The rAAV virion of any of Embodiments 1-37, wherein the expression cassette comprises 1, 2, 3, or 4 enhancers.

Embodiment 39: The rAAV virion of any of embodiments 1 to 38, wherein the expression cassette comprises one or two introns.

Embodiment 40: The rAAV virion of any of embodiments 1 to 39, wherein the expression cassette comprises one or two WPRE sequences.

Embodiment 41: The rAAV virion of any of embodiments 1-40, wherein the expression cassette comprises one or two polyadenylation sequences.

Embodiment 42: The rAAV virion of any of embodiments 1-41, wherein the expression cassette comprises about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb or less.

Embodiment 43: The method of any of embodiments 1-42, wherein the expression cassette is about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, A rAAV virion comprising about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

Embodiment 44: The method of any one of embodiments 1-3, wherein the expression cassette is at least 75%, 80%, 90%, 95%, 96%, 97 with any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. %, 98%, 99%, or 100% sequence identity, and optionally does not have a sequence or sequences encoding a DWORF (having an open reading frame or open reading frames encoding a DWORF) ), rAAV virions. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least 75%, 80%, 90% identical to a sequence within either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75. , 95%, 96%, 97%, 98%, 99%, or 100% identity, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) Replaced with a sequence or sequences encoding a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides).

Embodiment 45: The method of any of embodiments 1-3, wherein the expression cassette comprises either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75, and optionally does not have a sequence or sequences encoding DWORF (DWORF (having no open decoding frame or open decoding frames encoding), rAAV virion. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence within either SEQ ID NO: 20-24 or SEQ ID NO: 45-75, wherein encoding DWORF The sequence or sequences (open reading frame or open reading frames encoding DWORF) may be a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as those listed in Table 2b, which also provides sequences of such polypeptides). replaced by a sequence or sequences encoding any one of the polypeptides.

Embodiment 46: The method of any one of embodiments 1 to 3, wherein the expression cassette is selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60. A sequence or sequences comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one, and optionally encoding DWORF rAAV virions without (without an open decoding frame or open decoding frames encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and a sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60, wherein the sequence or sequence encoding DWORF (open reading frame or open reading frames encoding a DWORF) can be a polypeptide other than a DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides). replaced by a sequence or sequences encoding one).

Embodiment 47: The method of any one of embodiments 1 to 3, wherein the expression cassette is selected from SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60. A rAAV virion comprising either one, and optionally not having a sequence or sequences encoding a DWORF (having no open reading frame or open reading frames encoding a DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) is a polypeptide other than DWORF (e.g., a polypeptide described herein). replaced with a sequence or sequences encoding any one of the polypeptides (e.g., any one of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides).

Embodiment 48: The method of any one of embodiments 1-3, wherein the expression cassette is at least 75%, 80%, 90%, 95% identical to any one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58 , comprising a polynucleotide sequence that shares 96%, 97%, 98%, or 99% identity, and optionally does not have a sequence or sequences encoding a DWORF (an open reading frame or open reading frames encoding a DWORF) without), rAAV virions. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least 75%, 80% identical to at least one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58. , 90%, 95%, 96%, 97%, 98%, or 99% identity, wherein the sequence or sequences encoding DWORF (open reading frame or open reading frame encoding DWORF frames) is replaced with a sequence or sequences encoding a polypeptide other than DWORF (e.g., any one of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides) .

Embodiment 49: The method of any one of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, and optionally a sequence or sequence encoding DWORF. rAAV virions that do not have open decoding frames or open decoding frames that encode DWORF. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises at least one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, wherein The sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) may also provide a sequence of a polypeptide other than DWORF (e.g., any of the polypeptides described herein, e.g., such polypeptides). replaced with a sequence or sequences encoding any one of the polypeptides listed in Table 2b).

Embodiment 50: The method of any one of embodiments 1 to 3, wherein the expression cassette is at least 75% identical to any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75; Comprising polynucleotide sequences that share 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and optionally not having a sequence or sequences encoding a DWORF (encoding a DWORF) (having no open reading frame or open reading frames), rAAV virion. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75. a sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with one, wherein the sequence or sequences encoding a DWORF (DWORF encoding an open reading frame or open reading frames) encoding a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides) Replaced by a sequence or sequences that do.

Embodiment 51: The method of any of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75, and optionally A rAAV virion that does not have a sequence or sequences encoding DWORF (does not have an open reading frame or open reading frames encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75. Comprising, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) is a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as such polypeptide Replaced with a sequence or sequences encoding any one of the polypeptides listed in Table 2b, which also provides the sequence of.

Embodiment 52: The rAAV virion of any of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143).

Embodiment 53: The rAAV virion of any of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144).

Embodiment 54: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is a chimeric capsid protein.

Embodiment 55: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is an AAV5/AAV9 chimeric capsid protein.

Embodiment 56: The rAAV virion of any of embodiments 1-51, wherein the capsid protein is selected from any of SEQ ID NOs: 145-200.

Embodiment 57: An expression cassette comprising a polynucleotide sequence encoding a polypeptide operably linked to a promoter, wherein optionally the polypeptide is for expression in cardiac cells or tissues and/or for treating or preventing cardiac disease. An expression cassette for use in

Embodiment 58: The method of embodiment 57, wherein the polypeptide is selected from the group consisting of DWORF, JPH2, BAG3, CRYAB, lamin A isoform of LMNA, lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, selected from the DPII isoforms of DSP, DSG2, and JUP, wherein optionally the polypeptide is at least 90%, 95%, 96%, 97%, 98%, 99% identical to a sequence selected from the polypeptide sequences of Tables 2a and 2b. or an expression cassette that shares 100% sequence identity.

Embodiment 59: The expression cassette of embodiment 57 or 58, wherein the promoter is the chicken cTnT promoter.

Embodiment 60: The expression cassette of embodiment 59, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

Embodiment 61: The expression cassette of embodiment 59, wherein the chicken cTnT promoter comprises SEQ ID NO: 11.

Embodiment 62: The expression cassette of embodiment 57 or 58, wherein the promoter is the human cTnT promoter.

Embodiment 63: The expression cassette of embodiment 62, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 64: The expression cassette of embodiment 62, wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

Embodiment 65: The expression cassette of any one of embodiments 57-64, wherein the expression cassette further comprises one or more enhancers.

Embodiment 66: The expression cassette of embodiment 65, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

Embodiment 67: The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

Embodiment 68: The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78.

Embodiment 69: The expression cassette of embodiment 66, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

Embodiment 70: The expression cassette of embodiment 66, wherein the αMHC enhancer comprises SEQ ID NO:79.

Embodiment 71: The expression cassette of any of embodiments 57-70, wherein the expression cassette further comprises an intron.

Embodiment 72: The expression cassette of any of embodiments 57-70, wherein the intron is selected from a CMV intron and a chimeric intron.

Embodiment 73: The expression cassette of embodiment 72, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

Embodiment 74: The expression cassette of embodiment 72, wherein the CMV intron sequence comprises SEQ ID NO:80.

Embodiment 75: The expression cassette of embodiment 72, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

Embodiment 76: The expression cassette of embodiment 72, wherein the chimeric intron sequence comprises SEQ ID NO: 81.

Embodiment 77: The expression cassette of any one of embodiments 57-76, wherein the expression cassette further comprises a WPRE sequence.

Embodiment 78: The expression cassette of embodiment 77, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

Embodiment 79: The expression cassette of embodiment 77, wherein the WPRE sequence comprises SEQ ID NO:26.

Embodiment 80: The expression cassette of any one of embodiments 57-79, wherein the expression cassette further comprises a polyadenylation sequence.

Embodiment 81: The expression cassette of embodiment 80, wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and a SV40 polyadenylation sequence.

Embodiment 82: The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

Embodiment 83: The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence comprises SEQ ID NO: 27.

Embodiment 84: The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

Embodiment 85: The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence comprises SEQ ID NO: 28.

Embodiment 86: The expression cassette of any of embodiments 57-85, wherein the expression cassette is flanked by ITRs.

Embodiment 87: The expression cassette of embodiment 86, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15 .

Embodiment 88: The expression cassette of embodiment 86, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

Embodiment 89: The expression cassette of embodiments 57-88, comprising a single promoter.

Embodiment 90: The expression cassette of embodiments 57-88, comprising two promoters.

Embodiment 91: The expression cassette of any one of embodiments 57-90, comprising a single copy of the sequence encoding the polypeptide.

Embodiment 92: The expression cassette of any one of embodiments 57-90, comprising two copies of the sequence encoding the polypeptide.

Embodiment 93: The expression cassette of any of embodiments 57-92, comprising 1, 2, 3, or 4 enhancers.

Embodiment 94: The expression cassette of any of embodiments 57-93, comprising 1 or 2 introns.

Embodiment 95: The expression cassette of any one of embodiments 57-94, comprising one or two WPRE sequences.

Embodiment 96: The expression cassette of any one of embodiments 57-95, comprising one or two polyadenylation sequences.

Embodiment 97: The expression cassette of any of embodiments 57-96, comprising up to about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb.

Embodiment 98: The method of any one of embodiments 57-97, wherein the , an expression cassette comprising about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

Embodiment 99: The method of embodiment 57 or 58, wherein any of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75 and at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99 An expression cassette comprising polynucleotide sequences that share %, or 100% identity, and optionally not having a sequence or sequences encoding DWORF (having no open reading frame or open reading frames encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least 75%, 80%, 90% identical to the sequence of either SEQ ID NO: 20-24 or SEQ ID NO: 45-75. , 95%, 96%, 97%, 98%, 99%, or 100% identity, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) Replaced with a sequence or sequences encoding a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides).

Embodiment 100: The method of Embodiment 57 or 58, comprising a polynucleotide sequence of either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75, and optionally not having a sequence or sequences encoding DWORF (encoding DWORF) (having an open reading frame or no open reading frames), an expression cassette. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises either SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75, wherein the sequence encoding DWORF or The sequences (open reading frame or open reading frames encoding DWORF) may be polypeptides other than DWORF (e.g., any of the polypeptides described herein, e.g., one of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides). Replaced by the sequence or sequences encoding any one).

Embodiment 101: The method of embodiment 57 or 58, wherein any one of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60 and at least 75% , comprising polynucleotide sequences sharing 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and optionally not having a sequence or sequences encoding a DWORF (encoding a DWORF (having an open reading frame or no open reading frames), an expression cassette. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: A sequence or sequences that share at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 59 and SEQ ID NO: 60, wherein encoding DWORF (The open reading frame or open reading frames encoding DWORF) refers to a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides. ) is replaced by the sequence or sequences encoding.

Embodiment 102: The polynucleotide sequence of embodiment 57 or 58, wherein the polynucleotide sequence of any one of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60 An expression cassette comprising, and optionally not having a sequence or sequences encoding DWORF (not having an open reading frame or open reading frames encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) is a polypeptide other than DWORF (e.g., as described herein). Replaced with a sequence or sequences encoding any one of the polypeptides described, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides.

Embodiment 103: The method of embodiment 57 or 58, which is at least 75%, 80%, 90%, 95%, 96%, 97% with any one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58 , an expression cassette comprising polynucleotide sequences that share 98%, or 99% identity, and optionally not having a sequence or sequences encoding DWORF (having no open reading frame or open reading frames encoding DWORF) . In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least 75%, 80% identical to at least one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58. , 90%, 95%, 96%, 97%, 98%, or 99% identity, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) Replaced with a sequence or sequences encoding a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides).

Embodiment 104: The method of Embodiment 57, wherein the (having no open reading frame or open reading frames), expression cassette. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises at least one of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, wherein The sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) may also provide a sequence of a polypeptide other than DWORF (e.g., any of the polypeptides described herein, e.g., such polypeptides). replaced with a sequence or sequences encoding any one of the polypeptides listed in Table 2b).

Embodiment 105: The method of embodiment 57 or 58, wherein any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75 is at least 75%, 80%, 90%, comprising a polynucleotide sequence that shares 95%, 96%, 97%, 98%, or 99% identity, and optionally does not have a sequence or sequences encoding a DWORF (an open reading frame or open reading frame encoding a DWORF) (without frames), expression cassette. In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is at least SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75. A sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences or sequences encoding a DWORF ( an open reading frame or open reading frames) that encodes a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as any of the polypeptides listed in Table 2b, which also provides sequences of such polypeptides) Replaced by sequence or sequences.

Embodiment 106: The sequence of embodiment 57 or 58, comprising any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75, and optionally encoding DWORF. or an expression cassette that has no sequences (no open reading frame or open reading frames encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence is SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 74, and SEQ ID NO: 75. Comprising, wherein the sequence or sequences encoding DWORF (the open reading frame or open reading frames encoding DWORF) is a polypeptide other than DWORF (e.g., any of the polypeptides described herein, such as such polypeptide Replaced with a sequence or sequences encoding any one of the polypeptides listed in Table 2b, which also provides the sequence of.

Embodiment 107: The expression cassette of any one of embodiments 99-106, wherein the expression cassette does not comprise a 5' inverted terminal repeat and a 3' inverted terminal repeat of the polynucleotide sequence.

Embodiment 108: A pharmaceutical composition comprising the rAAV virion of any one of Embodiments 1 to 56 and a pharmaceutically acceptable carrier, or a vector comprising the expression cassette of any of Embodiments 57 to 107 and pharmaceutically A pharmaceutical composition comprising an acceptable carrier.

Embodiment 109: A kit comprising the pharmaceutical composition of embodiment 108.

Embodiment 110: A method of increasing polypeptide expression in a cell, said method comprising the rAAV virion of any of Embodiments 1-56, the vector comprising the expression cassette of any of Embodiments 57-107, or Embodiment 108. A method comprising contacting the cell with a composition of

Embodiment 111: The method of embodiment 110, wherein the cells are cardiac cells.

Embodiment 112: The method of embodiment 111, wherein the cells are cardiomyocytes.

Embodiment 113: The method of any one of embodiments 110-112, wherein polypeptide expression is increased by about 1.5-fold to 150-fold.

Embodiment 114: The method of any of embodiments 110-113, wherein the contacting occurs in vitro.

Embodiment 115: The method of any of embodiments 110-113, wherein the contacting occurs in vivo.

Embodiment 116: A method of increasing polypeptide expression in a tissue, said method comprising the rAAV virion of any one of embodiments 1-56, the vector comprising the expression cassette of any of embodiments 57-107, or embodiment 108. A method comprising contacting the tissue with a composition of

Embodiment 117: The method of embodiment 116, wherein the tissue is cardiac tissue.

Embodiment 118: The method of embodiment 116 or 117, wherein polypeptide expression is increased by about 1.5-fold to 150-fold.

Embodiment 119: The method of any of embodiments 116-118, wherein the contacting occurs in vitro.

Embodiment 120: The method of embodiment 116 or 118, wherein the contacting occurs in vivo.

Embodiment 121: A method of increasing polypeptide expression in an organ, said method comprising the rAAV virion of any one of embodiments 1-56, a vector comprising the expression cassette of any of embodiments 57-107, or embodiment 108. A method comprising contacting the organ with a composition of

Embodiment 122: The method of embodiment 121, wherein the organ is the heart.

Embodiment 123: The method of embodiment 122, wherein the patient has a heart condition or is at risk of developing a heart condition.

Embodiment 124: The method of embodiment 122 or 123, wherein the heart has a reduced or borderline ejection fraction.

Embodiment 125: The method of embodiment 122 or 123, wherein the heart has a normal ejection fraction.

Embodiment 126: The method of any one of embodiments 122-125, wherein the heart comprises a genetic mutation associated with heart disease.

Embodiment 127: The method of embodiment 126, wherein the genetic mutation is a PLN mutation.

Embodiment 128: The method of any of embodiments 121-127, wherein the heart has low or undetectable levels of polypeptide expression compared to a healthy heart.

Embodiment 129: The method of any of embodiments 121-128, wherein polypeptide expression is increased by about 1.5-fold to 150-fold.

Embodiment 130: The method of any of embodiments 121-129, wherein the contacting occurs in vitro.

Embodiment 131: The method of any of embodiments 121-129, wherein the contacting occurs in vivo.

Embodiment 132: A method of increasing polypeptide expression in a subject, comprising the rAAV virion of any one of embodiments 1-56, a vector comprising the expression cassette of any of embodiments 57-107, or embodiment 108. A method comprising administering a composition of to the subject.

Embodiment 133: The method of embodiment 132, wherein the subject is an animal.

Embodiment 134: The method of embodiment 132, wherein the subject is a human.

Embodiment 135: The method of any one of embodiments 132-134, wherein polypeptide expression is increased in the heart of the subject.

Embodiment 136: The method of any one of embodiments 132-135, wherein the subject has heart disease or is at risk for heart disease.

Embodiment 137: The method of any of embodiments 132-136, wherein the subject has borderline or reduced ejection fraction.

Embodiment 138: The method of any of embodiments 132-136, wherein the subject has a normal ejection fraction.

Embodiment 139: The method of any one of embodiments 132-138, wherein the subject has a genetic mutation associated with heart disease.

Embodiment 140: The method of embodiment 139, wherein the genetic mutation is a PLN mutation.

Embodiment 141: The method of any of embodiments 132-140, wherein the subject has low or undetectable levels of polypeptide expression compared to a healthy subject.

Embodiment 142: A method of treating a heart disease or disorder in a subject in need thereof, comprising comprising the rAAV virion of any one of embodiments 1-56, the expression cassette of any of embodiments 57-107 A method comprising administering to the subject a vector comprising, or the composition of embodiment 108.

Embodiment 143: The method of embodiment 142, wherein the subject has a heart disease or disorder.

Embodiment 144: The method of embodiment 142, wherein the subject is at risk of developing a heart disease or disorder.

Embodiment 145: The method of any one of embodiments 142-144, wherein the heart disease or disorder is cardiomyopathy.

Embodiment 146: The method of any one of embodiments 142-144, wherein the heart disease or disorder is dilated cardiomyopathy.

Embodiment 147: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is myocardial infarction.

Embodiment 148: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is chronic myocardial infarction.

Embodiment 149: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is acute myocardial infarction.

Embodiment 150: The method of any one of embodiments 142-149, wherein the subject has a genetic risk allele for a heart disease or disorder.

Embodiment 151: The method of any one of embodiments 142-150, wherein the genetic risk allele comprises a mutation to the PLN gene.

Embodiment 152: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN promoter mutation.

Embodiment 153: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN ^L39stop mutation.

Embodiment 154: The method of embodiment 151, wherein the mutation to the PLN gene is an RC9 mutation.

Embodiment 155: The method of embodiment 151, wherein the mutation to the PLN gene is an R9L mutation.

Embodiment 156: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN gene duplication.

Embodiment 157: The method of embodiment 151, wherein the mutation to the PLN gene is an R14del mutation.

Embodiment 158: The method of any one of embodiments 142-157, wherein the cardiac disease or disorder is reduced ejection fraction (HFrEF).

Embodiment 159: The method of any of embodiments 142-157, wherein the cardiac disease or disorder has preserved ejection fraction (HFpEF).

Embodiment 160: The method of any one of embodiments 142-159, wherein the method causes expression of the polypeptide in the heart of the subject.

Embodiment 161: The method of any one of embodiments 142-160, wherein the method causes expression of the polypeptide in cardiomyocytes.

Embodiment 162: The method of any one of embodiments 142-161, wherein the method does not result in detectable expression of the polypeptide in muscles of the subject other than the heart.

Embodiment 163: The method of any one of embodiments 142-162, wherein the method does not result in detectable expression of the polypeptide in the liver of the subject.

Embodiment 164: The method of any one of embodiments 142-163, wherein the method does not result in detectable expression of the polypeptide in cardiac fibroblasts.

Embodiment 165: The method of any one of embodiments 142-164, wherein the method improves one or more measures of cardiac function, optionally fractional shortening and/or left ventricular internal dimension (LVID).

Embodiment 166: The method of any one of embodiments 142-165, wherein an improvement in cardiac function is observed from week 2 to week 24.

Embodiment 167: The method of any one of embodiments 142-166, wherein the method reduces cardiac remodeling.

Embodiment 168: The method of any one of embodiments 142-166, wherein the method corresponds to a decrease in polypeptide expression in a subject suffering from or at risk of heart disease.

Embodiment 169: The method of any one of embodiments 142-168, wherein the administering is systemic administration.

Embodiment 170: The method of embodiment 169, wherein the systemic administration is selected from intravenous or intraluminal injection.

Embodiment 171: The method of embodiment 169 or 170, wherein the rAAV is administered as a unit dose.

Embodiment 172: The method of embodiment 171, wherein the unit dose is less than or equal to about 3Х10 ¹⁴ vg/kg, less than or equal to about 2Х10 ¹⁴ vg/kg, less than or equal to about 1Х10 ¹⁴ vg/kg, less than or equal to about 9Х10 ¹³ vg/kg, or less than or equal to about 8Х10 ¹³ vg. /kg or less, about 7Х10 ¹³ vg/kg or less, about 6Х10 ¹³ vg/kg or less, about 5Х10 ¹³ vg/kg or less, about 4Х10 ¹³ vg/kg or less, about 3Х10 ¹³ vg/kg or less, about 2Х10 ¹³ vg/kg or less or less, or comprising less than or equal to about 1Х10 ¹³ vg/kg.

Embodiment 173: A method of alleviating one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising the rAAV virion of any one of embodiments 1-56, any of embodiments 57-107. A method comprising administering a vector comprising an expression cassette, or the composition of embodiment 108.

Embodiment 174: A method of improving one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising the rAAV virion of any one of embodiments 1-56, any of embodiments 57-107 A method comprising administering a vector comprising an expression cassette, or the composition of embodiment 108.

Embodiment 175: A method of preventing one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising the rAAV virion of any one of embodiments 1-56, any of embodiments 57-107 A method comprising administering a vector comprising an expression cassette, or the composition of embodiment 108.

Embodiment 176: An expression cassette comprising a polynucleotide:

i. one or more promoters;

ii. Optionally one or more enhancers;

iii. optionally one or more introns;

iv. One or more transgenes (optionally, wherein the one or more transgenes encode one or more polypeptides for use in treating or preventing heart disease);

v. Optionally one or more WPRE sequences; and

vi. An expression cassette, optionally comprising one or more polyadenylation sequences, p(A).

Implementation 177: The method of implementation 176, wherein the 5' to 3' arrangement of the elements is:

(i) 5'-promoter-transgene-WPRE-p(A)-3';

(ii) 5'-promoter-intron-transgene-WPRE-p(A)-3';

(iii) 5'-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A);

(iv) 5'-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

(v) 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

(vi) 5'-Enhancer-Enhancer-Promoter-Transgene-WPRE-p(A)-3';

(vii) 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-3';

(viii) 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';

ix) 5'-Enhancer-Promoter-Intron-Transgene-WPRE-p (A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';

(x) 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

(xi) 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

(xii) 5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; and

(xiii) 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3';

wherein, optionally, the transgene is an expression cassette encoding a polypeptide for use in treating or preventing heart disease.

Embodiment 178: The method of embodiment 176 or embodiment 177, wherein the transgene is a polynucleotide having an arrangement of elements from 5' to 3' comprising 5'-promoter-transgene-WPRE-p(A)-3'. An expression cassette having an increased expression level compared to a second expression cassette comprising.

Embodiment 179: The expression cassette of embodiment 178, wherein the level of increased expression is from about 1.5-fold to about 150-fold compared to the second expression cassette.

Embodiment 180: A recombinant adeno-associated virus (rAAV) virion, comprising a viral genome comprising a capsid protein and the expression cassette of any of embodiments 176 to 179, wherein the expression cassette is flanked by inverted terminal repeats. A recombinant adeno-associated virus (rAAV) virion.

Embodiment 181: The rAAV of embodiment 180, wherein the expression cassette comprises a transgene, wherein the transgene encodes a polypeptide for use in treating or preventing heart disease or alleviating symptoms associated with heart disease.

Embodiment 182: The rAAV of embodiment 180 or embodiment 181, wherein the capsid protein is selected from any one of SEQ ID NOs: 145-200.

Example

Example 1: Novel expression cassette

The purpose of this study was to evaluate several engineered expression cassettes for their ability to express transgenes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and mouse models.

The human DWORF (hDWORF) polynucleotide (SEQ ID NO: 33) was inserted into an expression cassette designed to induce strong cardiomyocyte-specific expression while maintaining a total size of 3-4.7 kbp. Three native expression cassettes were generated, pCR-HD1 (SEQ ID NO: 22), pCR-HD2 (SEQ ID NO: 23), and pCR-HD3. Figure 1 shows exemplary embodiments of each expression cassette, comprising repeats of the cardiac troponin T promoter (see Table 3), WPRE (SEQ ID NO: 26) and BGH poly(A) signal (SEQ ID NO: 27). To assess the expression of the transgenes encoded by each expression cassette in hiPSC-CMs, the DWORF transgenes were cloned into CR-G1, CR-G2, and CR-G2, corresponding to pCR-HD1, pCR-HD2, and pCR-HD3, respectively. GFP was replaced in each expression cassette, designated CR-G3. CR-G1, CR-G2, and CR-G3 expression cassettes were packaged into rAAV virions using the AAV6 capsid (SEQ ID NO: 39) protein. Cells were infected with each rAAV virion containing the expression cassette variant at an MOI of 50,000, and GFP expression was quantified by flow cytometry. Figure 2 shows that pCR-G1, pCR-G2, and pCR-G3 expression cassettes showed high levels of expression in hiPSC-CMs. Surprisingly, the double transgene construct showed lower expression, likely due to homologous recombination between tandem repeat sequences. Next, expression cassettes containing hDWORF, pCR-HD1, and pCR-HD2 were evaluated in a wild-type CD1 mouse model. Each cassette was packaged into rAAV virions using the AAV9 capsid protein. rAAV virions (5Х10 ¹¹ vg) were administered systemically to wild-type CD1 mice (N=2) by retro-orbital injection. Two weeks after injection, animals were sacrificed and hDWORF expression in the heart was assessed. Figure 3 shows that the pCR-HD2 expression cassette resulted in an approximately 2-fold increase in hDWORF expression. We conclude that increasing the length of the canonical hTNNT2 promoter from 600 bp to 1.5 kb, increasing the total vector size from 1.9 kb to 3.2 kb, and/or inserting an intron surprisingly doubles gene expression.

Example 2: Evaluation in hiPSC-CM

The purpose of this study was to evaluate the ability of chimeric capsid proteins to facilitate infection of hiPSC-CMs with rAAV virions containing expression cassettes encoding HA-tagged hDWORF proteins and chimeric capsid proteins.

The expression cassette encoding the HA-tagged hDWORF protein was packaged into rAAV virions using one of five chimeric capsid proteins. The chimeric capsid proteins tested were CR9-01 (SEQ ID NO: 29), CR9-10 (SEQ ID NO: 19), CR9-14 (SEQ ID NO: 30), TN44-07 (SEQ ID NO: 31), or TN47-10 (SEQ ID NO: 18). ), and AAV9 capsid protein (SEQ ID NO: 16) was used as a control. Wild-type hiPSC-CMs were infected at an MOI of 3,900. At day 5 post infection, cells were fixed and stained for SERCA2a and HA-tagged hDWORF. Stained cells were imaged ( Figure 4B ), quantified ( Figure 4A ), and expression was normalized to AAV9 using a cellular imaging device. Figure 4A shows HA-tagged hDWORF expression in hiPSC-CM cell cultures infected with rAAV virions containing CR9-01, CR9-10, CR9-14, or TN44-07 chimeric capsid proteins. Each of these capsids performed equally well or better than the AAV9 capsid protein in delivering functional expression cassettes.

Example 3: Evaluation in Murine Model

The purpose of this study was to evaluate the ability of the chimeric capsid protein to facilitate infection of heart tissue of wild-type mice with rAAV virions containing an expression cassette encoding the HA-tagged hDWORF protein and the chimeric capsid protein.

The expression cassette encoding the HA-tagged hDWORF protein was packaged into rAAV virions using one of five chimeric capsid proteins. The chimeric capsid proteins tested were CR9-01 (SEQ ID NO: 29), CR9-10 (SEQ ID NO: 19), CR9-14 (SEQ ID NO: 30), TN44-07 (SEQ ID NO: 31), or TN47-10 (SEQ ID NO: 18). ), and AAV9 capsid protein (SEQ ID NO: 16) was used as a control. rAAV virions were delivered by retro-orbital injection to wild-type mice at a dose of 5x10 ¹¹ vg/mouse (N=3). At day 14 after injection, animals were sacrificed and heart tissue was harvested for RNA and protein analysis. Figure 5A shows hDWORF RNA expression in heart tissue infected with the indicated capsids. All expression levels were normalized to that of the AAV9 group. The results show that in vivo delivery of rAAV virions packaged with CR9-01, CR9-10, CR9-14, TN44-07, and TN47-10 each increased hDWORF expression compared to parental AAV9. rAAV virions containing CR9-10 and TN47-10 capsids showed the highest level of expression compared to the other capsids tested (approximately 9-fold and approximately 11-fold increase, respectively). Figure 5B shows that hDWORF protein expression corroborates RNA expression levels in heart tissue, indicating that hDWORF RNA was translated into protein in infected cells and that the modified capsid dramatically increased protein expression.

Example 4: PLN-R14 ^Δ/Δ DWORF gene therapy in models

The purpose of this study was to evaluate the ability of rAAV virions containing an expression cassette encoding the mDWORF protein to reduce symptoms associated with cardiomyopathy in mice carrying the PLN-R14 deletion mutation.

Transgenic mice expressing a homozygous PLN gene carrying an arginine 14 deletion (PLN-R14Δ ^/Δ ) recapitulate human cardiomyopathy and display similar histopathological abnormalities and early death (Haghighi K et al., Proc. Natl Acad. Sci. USA 103:1388-1393 (2006)). In particular, transgenic mice can be used as a model for dilated cardiomyopathy. This model is significantly more severe than the MLP ^−/− knockout and is clinically relevant because there are no known patients with MLP knockout cardiomyopathy.

In this study, 3-week-old homozygous PLN-R14del animals were incubated with either rAAV virions consisting of the pCR-MD1 expression cassette (SEQ ID NO: 20) and the AAV9 capsid protein or Hank's Balanced Salt Solution (HBSS) as a placebo control. was injected. PLN-R14 ^Δ/Δ mice were administered rAAV virions by retro-orbital injection at doses ranging from 5Х10 ¹² vg/kg to 5Х10 ¹³ vg/kg (N=7 to 8/group). Ejection fraction and fractional shortening were assessed by echocardiography as markers of cardiac function at 6.5 weeks of age. Figure 6A shows dose-dependent improvement in ejection fraction compared to placebo control (p = 0.035). Figure 6B shows dose-dependent improvement in fraction shortening compared to placebo control (p = 0.028).

Taken together, these results indicate that systemic administration of rAAV virions expressing DWORF improves cardiac function in an animal model of cardiomyopathy. Observation of these effects in mice treated at 3 weeks of age demonstrates that this gene therapy can treat and prevent the development of hypertrophic cardiomyopathy (HCM).

Example 5: rAAV-mediated DWORF expression

The expression cassette was packaged into rAAV virions along with the AAV9 capsid and injected into 4-week-old wild-type C57Bl6 mice at a dose of 5Х10 ¹³ vg/kg (N=4). Mouse hearts were collected 3 weeks after injection. DWORF protein expression was determined and quantified by Western blot ( Figure 8C , Table 10). Transgenic mice with high DWORF expression levels (Tg-dwarf) were used as positive controls.

The results show that combinations of specific promoters and enhancers and their orientation in the expression cassette result in different levels of DWORF expression. The specific expression cassettes and associated elements used in this example, and their orientation, are presented in Figures 7A-7C . DWORF expression increased with the addition of elements compared to the promoter alone, but the degree of increase in DWORF expression could not be predicted based on element and orientation alone.

Figure 8A shows the strategy for assessing the expression of the transgene (DWORF) in cardiomyocytes of untreated mice. At 3 weeks after injection, animals were sacrificed and DWORF expression in the heart was assessed. Figure 8b shows expression analysis results for 16 AAV constructs with various regulatory elements and arrangements to increase CM-selective expression of the transgene (DWORF). In particular, Figure 8B depicts the expression of DWORF in wild-type C57BL6 mouse cardiomyocytes harvested 3 weeks after retro-orbital injection of the AAV9:DWORF vector, as assessed by Western blot; Here the lower Western blot panel shows the expression of GAPDH as a positive control. Figure 8C shows that combinations of specific promoters and enhancers and their orientation in expression cassettes result in different levels of transgene expression. Transgenic mice with high DWORF expression levels (Tg-dwarf) were used as positive controls in this example.

For each expression cassette, DWORF expression was normalized to the level observed for the pCRmD1 expression cassette with only the human cTnT promoter without added enhancer elements. Addition of the ACTC1 enhancer (pHZ15) or the αMHC enhancer (pHZ17) increased DWORF expression about 3-fold to about 4-fold compared to pCRmD1. Addition of the CMV intron (pHZ20) was observed to increase DWORF expression approximately 5-fold. The combination of promoter, single enhancer, and intron (pHZ22 and pHZ23) did not significantly increase DWORF expression compared to any element alone. The combination of both promoter and enhancer only slightly increased DWORF expression by approximately 6-8 fold compared to the promoter alone. Surprisingly, the combination of enhancer, promoter, and intron all increased DWORF expression approximately 10- to 16-fold. Interestingly, enhancers in the 5' to 3' direction play a fine-tuning role in regulating protein expression. Orienting ACTC1 enhancer 5' to a second enhancer (pHZ16 or pHZ21) appears to increase DWORF expression compared to orienting ACTC1 enhancer 3' to a second enhancer (pHZ18 or pHZ19). Inclusion of a codon-optimized DWORF transgene (pHZ24) also increased expression.

Addition of various regulatory elements significantly increased DWORF expression, but addition of more copies of the transgene (pHZ25, pHZ33, pHZ34, pHZ69, pHZ72 and pHZ75) caused an unexpected synergistic effect on DWORF levels. We used different enhancers, promoters, introns, and codon-optimized DWORFs in each copy to avoid homologous recombination between tandem repeat sequences. The double copy vector has approximately 40 to 140 times more expression than DWORF under transcriptional control of the promoter alone. Surprisingly, the orientation of the two copies plays an unexpected role in regulating gene expression. For example, the tail-to-tail orientation showed the best expression, followed by the head-to-head and head-to-tail arrangements. The results also suggest that different arrangements of the two copies of the transgene can be used to fine-tune transgene expression.

Example 6: Effect of DWORF Expression on Ejection Fraction

The effect of rAAV-mediated DWORF expression on ejection fraction was determined in the MLP knockout (MLP-KO) dilated cardiomyopathy (DCM) model. Three of the rAAV virions were tested, including a virion with a vector genome with single copy expression cassettes pHZ19 and pHZ21, and a virion with a double copy expression cassette pHZ34. MLP-KO mice were administered either 5Х10 ¹³ vg/kg of pHZ19, 5Х10 ¹³ vg/kg of pHZ21, or 1Х10 ¹³ vg/kg of pHZ34. Virions were delivered by retro-orbital injection at 6 weeks of age, when mice exhibited moderate heart failure. Cardiac function was assessed by echocardiography at 3, 6, 9, 12, 16, and 20 weeks after treatment. As shown in Figure 9 , pHZ21 at a dose of 5Х10 ¹³ vg/kg significantly improved ejection fraction (14.4%) up to 16 weeks after virus injection compared to the HBSS group. Although pHZ21 has only 1.6 times more DWORF expression than pHZ19, this moderate improvement in expression appears to have a very profound effect in improving cardiac function. Although the dosage of pHZ34 is 5 times lower than pHZ19 and pHZ21, it achieved similar improvement as pHZ21 and better improvement than pHZ19, highlighting the importance of DWORF expression.

Three DWORF expression cassettes were tested in another well-characterized DCM model, the BAG3 cardiac conditional knockout (BAG3-cKO) model. The DWORF expression cassette in this model included one single copy vector, pHZ21, and two double copy vectors, pHZ72 and pHZ75. BAG3-cKO mice were administered 5Х10 ¹³ vg/kg of AAV9-pHZ21, AAV9-pHZ72, and AAV9-pHZ75. Virions were delivered by retro-orbital injection to mice at 8 weeks of age, when moderate heart failure had already occurred. Heart function was evaluated by echocardiography at 3 and 6 weeks after treatment. As shown in Figure 10 , pHZ72 at a dose of 5Х10 ¹³ vg/kg substantially improved the ejection fraction (7.4%) by week 6 after virus injection compared to the HBSS group, indicating that this optimized DWORF gene therapy is effective in this model. suggests that it can improve heart function.

Example 7: Effect of DWORF Construct in Severe MLP-KO DCM Mouse Model

The purpose of this study was to test how optimized DWORF vectors can improve cardiac function and exercise capacity in a well-characterized MLP knockout (MLP-KO) dilated cardiomyopathy model.

Figure 11A is a schematic diagram of the study design. Three of the rAAV virions were tested, including a virion with a vector genome with single copy expression cassettes pHZ19 and pHZ21, and a virion with a double copy expression cassette pHZ34. MLP-KO mice were administered either 5Х10 ¹³ vg/kg of pHZ19, 5Х10 ¹³ vg/kg of pHZ21, or 1Х10 ¹³ vg/kg of pHZ34. Virions were delivered by retro-orbital injection at 6 weeks of age, when mice exhibited moderate heart failure. Heart function was evaluated by echocardiography at 3-4 weeks and up to 24 weeks after treatment. MLP-KO mice have limited motor skills compared to their wild-type littermates. To assess how much DWORF gene therapy can improve exercise performance in MLP-KO mice, mice were run on a rodent treadmill and their running duration was monitored at 26 weeks.

As shown in Figures 11b and 11c , pHZ21 at a dose of 5Х10 ¹³ vg/kg significantly improved ejection fraction (14.3%) up to 24 weeks after virus injection compared to the HBSS group. Although pHZ21 has only 1.6 times more DWORF expression than pHZ19, this moderate improvement in expression appears to have a very profound effect in improving cardiac function. Although the dosage of pHZ34 is 5 times lower than pHZ19 and pHZ21, it achieved similar improvement as pHZ21 and better improvement than pHZ19, highlighting the importance of DWORF expression. Figures 11D and 11E show that MLP-KO mice have limited motor abilities compared to their wild-type littermates. pHZ21, the best DWORF vector to improve ejection fraction, also significantly improved exercise capacity, including running distance and time to exhaustion, in the MLP KO DCM mouse model at 26 weeks post-treatment compared to saline controls.

Overall, the study indicates that AAV-delivered DWORF alleviated systolic dysfunction and improved exercise capacity in this MLP-KO DCM model. The AAV:DWORF cassette expressed higher levels of DWORF, supporting the most significant degree of efficacy that persisted through week 24. These results indicate that DWORF gene therapy can be used to normalize calcium homeostasis and limit disease progression.

Example 8: Tolerability of DWORF gene therapy in untreated mice

The aim of this study was to evaluate the tolerability of DWORF gene therapy in untreated mice. Figure 12A is a schematic diagram of the study design. Untreated mice were administered HBSS saline control or AAV9:pHZ21 vector in two doses: 5Х10 ¹³ vg/kg and 2Х10 ¹⁴ vg/kg. Virus was delivered via intraperitoneal (IP) injection on postnatal day 3 (P3). Heart function was assessed by echocardiography 4 weeks after virus injection.

Figure 12b shows that AAV9:pHZ21 is well tolerated up to 2Х10 ¹⁴ vg/kg in wild type mice. There are no differences in body weight, ejection fraction, heart rate, and left ventricular mass (LV mass) for the group administered AAV9:pHZ21 compared to the saline control group.

Incorporation by reference

Various references, such as patents, patent applications, and patent publications, are incorporated herein by reference, the disclosures of which are hereby incorporated by reference in their entirety. Additionally, all references mentioned herein are incorporated by reference to specifically disclose and describe the methods and/or materials with which the publications are cited.

Claims (101)

As an expression cassette comprising a polynucleotide sequence:
i) one or more promoters, optionally said one or more promoters being cardiac-specific promoters; and
ii) one or more copies of a transgene, wherein the transgene encodes a polypeptide for treating or preventing heart disease or alleviating symptoms associated with heart disease;
wherein the expression cassette is:
iii) a polynucleotide sequence further comprising one or more enhancers;
iv) one or more copies of the transgene, wherein there are at least two copies of the transgene;
v) a polynucleotide sequence further comprising one or more introns; and/or
vi) an expression cassette that is codon optimized and comprises one or more of at least one copy of one or more copies of said transgene. The expression cassette of claim 1 , wherein the polynucleotide sequence comprises one or more enhancers, optionally wherein the one or more enhancers are cardiac-specific enhancers. 3. The method of claim 1 or 2, wherein the one or more copies of the transgene are two copies of the transgene, optionally the one or more promoters are two promoters, and further optionally, the two copies of the transgene are identical. No, expression cassette. The expression cassette according to any one of claims 1 to 3, wherein the polynucleotide sequence further comprises one or more introns, wherein optionally the one or more introns improve the efficiency of transgene expression. 5. The method of any one of claims 1 to 4, wherein at least one copy of the one or more copies of the transgene is codon optimized, and optionally, one or more copies of the transgene are two copies of the transgene and One copy of the expression cassette is not codon optimized. The method of any one of claims 1 to 5, wherein the polyadenylation sequence is:
i) one or more post-transcriptional regulatory elements (“PTREs”), optionally wherein the one or more post-transcriptional regulatory elements are WPREs; and/or
ii) an expression cassette, further comprising one or more polyadenylation sequences (“p(A)”). According to any one of claims 1 to 6,
(i) 5'-promoter-intron-transgene-PTRE-p(A)-3';
(ii) 5'-promoter-transgene-PTRE-p(A)-promoter-transgene-PTRE-p(A);
(iii) 5'-Enhancer-Promoter-Transgene-PTRE-p(A)-3';
(iv) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-3';
(v) 5'-Enhancer-Enhancer-Promoter-Transgene-PTRE-p(A)-3';
(vi) 5'-Enhancer-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-3';
(vii) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-p(A)-Transgene-Intron-Promoter-Enhancer-3';
(viii) 5'-Enhancer-Promoter-Intron-Transgene-PTRE-p(A)-Enhancer-Promoter-Intron-Transgene-p(A)-3';
(ix) 5'-p(A)-PTRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';
(x) 5'-promoter-intron-transgene-PTRE-p(A)-p(A)-transgene-intron-promoter-3';
(xi) 5'-promoter-intron-transgene-PTRE-p(A)-promoter-intron-transgene-p(A)-3'; and
(xii) expression, comprising a 5' to 3' arrangement of elements selected from 5'-p(A)-PTRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3' cassette. The method according to any one of claims 1 to 7, wherein the transgene is a polynucleotide having an arrangement of elements comprising 5'-promoter-transgene-WPRE-p(A)-3' from 5' to 3'. An expression cassette having an increased expression level compared to an expression cassette comprising. 9. The method of claim 8, wherein the increased expression level is from about 1.5-fold to about 150-fold, optionally wherein the increased expression level is at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold. expression cassette, or at least 100-fold. 10. The expression cassette according to any one of claims 1 to 9, wherein at least one of the one or more promoters is a chicken cTnT promoter. 11. The expression cassette of claim 10, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. 11. The expression cassette of claim 10, wherein the chicken cTnT promoter comprises SEQ ID NO: 11. 13. The expression cassette according to any one of claims 1 to 12, wherein at least one of the one or more promoters is a human cTnT promoter. 14. The expression cassette of claim 13, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. 14. The expression cassette of claim 13, wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13. 16. The expression cassette of any one of claims 1 to 15, wherein the one or more enhancers are one or more cardiac specific enhancers. 17. The expression cassette of claim 16, wherein the one or more cardiac specific enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer. 18. The expression cassette of claim 17, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78. 18. The expression cassette of claim 17, wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78. 18. The expression cassette of claim 17, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. 18. The expression cassette of claim 17, wherein the αMHC enhancer comprises SEQ ID NO: 79. 22. The expression cassette according to any one of claims 1 to 21, wherein the intron is selected from CMV introns and chimeric introns. 23. The expression cassette of claim 22, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80. 23. The expression cassette of claim 22, wherein the CMV intron sequence comprises SEQ ID NO: 80. 23. The expression cassette of claim 22, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. 23. The expression cassette of claim 22, wherein the chimeric intron sequence comprises SEQ ID NO: 81. 27. The expression cassette of any one of claims 1 to 26, wherein the expression cassette comprises a WPRE sequence. 28. The expression cassette of claim 27, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26. 28. The expression cassette of claim 27, wherein the WPRE sequence comprises SEQ ID NO:26. 30. The expression cassette of any one of claims 1 to 29, wherein the expression cassette comprises a polyadenylation sequence. 31. The expression cassette of claim 30, wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence. 32. The expression cassette of claim 31, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. 32. The expression cassette of claim 31, wherein the BGH polyadenylation sequence comprises SEQ ID NO: 27. 32. The expression cassette of claim 31, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. 32. The expression cassette of claim 31, wherein the SV40 polyadenylation sequence comprises SEQ ID NO: 28. 36. The expression cassette of any one of claims 1-35, wherein the expression cassette is flanked by ITRs. 37. The expression cassette of claim 36, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. 37. The expression cassette of claim 36, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15. 39. The expression cassette of any one of claims 1-38, comprising a single promoter. 39. The expression cassette of any one of claims 1-38, comprising two promoters. 41. The expression cassette of any one of claims 1-40, comprising from about 1.9 kb to about 3.7 kb. 41. The expression cassette of any one of claims 1 to 40, comprising from about 2.5 kb to about 3.7 kb, optionally from about 2.8 kb to about 3.6 kb. 43. The method of any one of claims 1 to 42, wherein the transgene is DWORF, JPH2, BAG3, CRYAB, lamin A isoform of LMNA, lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DSP An expression cassette encoding a polypeptide selected from the DPI isoform of DSP, the DPII isoform of DSP, DSG2, and JUP. The method of any one of claims 1 to 43, wherein the transgene is SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: No. 217, SEQ ID No. 219, Seq. Expression cassettes that share 99%, or 100% sequence identity. 45. The method of any one of claims 1 to 44, wherein the polypeptide is SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, or SEQ ID NO: 230 and at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99 Expression cassettes that share %, or 100% sequence identity. 44. The expression cassette of any one of claims 1-43, wherein the transgene encodes a DWORF polypeptide. 47. The method of claim 46, wherein the transgene is at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 76, or SEQ ID NO: 77, Expression cassettes that share 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. 47. The method of claim 46, wherein the polypeptide is at least 75%, 80%, 90%, 95%, Expression cassettes that share 96%, 97%, 98%, 99%, or 100% sequence identity. 49. The expression cassette of any one of claims 43-48, wherein the transgene comprises a codon optimized nucleic acid sequence. According to any one of claims 1 to 49,
(i) 5'-human or chicken TnT promoter-chimeric intron-transgene-WPRE-p(A)-3';
(ii) 5'-first human or chicken TnT promoter-first copy of the transgene-WPRE-p(A)-second human or chicken TnT promoter-second copy of the transgene-WPRE-p(A) ( Optionally, wherein the first and second promoter sequences and the first and second copies of the transgene are in the same forward orientation;
(iii) 5'-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(iv) 5'-ACTC1e enhancer-heart-human TnT promoter-transgene-WPRE-bGHpA-3';
(v) 5'-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';
(vi) 5'-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(vii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(viii) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';
(ix) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-transgene-WPRE-bGHpA-3';
(x) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(xi) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(xii) 5'-human TnT promoter-transgene with codon optimized polynucleotide sequence-WPRE-bGHpA-3';
(xiii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);
(xiv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation);
(xv) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally, wherein the first transgene and the human TnT promoter are in reverse orientation and the second transgene and the chicken TnT promoter are in forward orientation);
(xvi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter- 3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);
(xvii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation); and
(xviii) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene and the human TnT promoter are in a reverse orientation and the second transgene and the chicken TnT promoter are in a forward orientation). cassette. According to clause 50,
(i) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(ii) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3';
(iii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);
(iv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation);
(v) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., codon optimized polynucleotide sequence having) -SV40pA-3' (optionally, wherein the first transgene and the human TnT promoter are in reverse orientation and the second transgene and the chicken TnT promoter are in forward orientation);
(vi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with codon optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter- 3' (optionally, wherein the first transgene and the human TnT promoter are in forward orientation and the second transgene and chicken TnT promoter are in reverse orientation);
(vii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene, human TnT promoter, second transgene and chicken TnT promoter are in forward orientation); and
(viii) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA- 3' (optionally, wherein the first transgene and the human TnT promoter are in a reverse orientation and the second transgene and the chicken TnT promoter are in a forward orientation). cassette. 52. The expression cassette according to any one of claims 1 to 51, wherein the expression cassette is a recombinant expression cassette. A recombinant vector comprising the expression cassette of any one of claims 1 to 52. A recombinant adeno-associated virus (rAAV) virion, comprising a capsid protein and a viral genome comprising the expression cassette of any one of claims 1 to 52, wherein the expression cassette is flanked by inverted terminal repeats (ITRs). Located on recombinant adeno-associated virus (rAAV) virions. 55. The rAAV virion of claim 54, wherein the ITR shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. 55. The rAAV virion of claim 54, wherein the ITR comprises one or more of SEQ ID NO: 14 and SEQ ID NO: 15. 57. The rAAV virion of any one of claims 54-56, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143). 57. The rAAV virion of any one of claims 54-56, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144). 57. The rAAV virion according to any one of claims 54 to 56, wherein the capsid protein is a chimeric capsid protein, 57. The rAAV virion according to any one of claims 54 to 56, wherein the capsid protein is an AAV5/AAV9 chimeric capsid protein, 57. The rAAV virion of any one of claims 54-56, wherein the capsid protein is selected from any of SEQ ID NOs: 145-200. A pharmaceutical composition comprising the vector of claim 53, or the rAAV virion of any one of claims 54 to 61, and a pharmaceutically acceptable carrier. A kit comprising the pharmaceutical composition of claim 62. 1. A method of increasing the expression of a polypeptide in a cardiac cell or cardiac tissue, comprising contacting said cell with the vector of claim 53, the rAAV virion of any of claims 54-61, or the composition of claim 62. Method, including. 65. The method of claim 64, wherein the heart cells are cardiomyocytes. 65. The method of claim 64, wherein the cardiac tissue is cardiac tissue. 67. The method of any one of claims 64-66, wherein polypeptide expression is increased by about 1.5-fold to 150-fold. 68. The method of any one of claims 64-67, wherein the contacting step occurs in vitro. 68. The method of any one of claims 64-67, wherein the contacting occurs in vivo. 1. A method of increasing expression of a polypeptide in a subject, comprising administering to the subject the vector of claim 53, the rAAV virion of any of claims 54-61, or the composition of claim 62, method. 71. The method of claim 70, wherein the subject is a mammal. 71. The method of claim 70, wherein the subject is a human. 73. The method of any one of claims 70-72, wherein polypeptide expression is increased in the heart of the subject. 74. The method of any one of claims 70-73, wherein the subject suffers from or is at risk for heart disease. 75. The method of any one of claims 70-74, wherein the subject has borderline or reduced ejection fraction. 75. The method of any one of claims 70-74, wherein the subject has a normal ejection fraction. 77. The method of any one of claims 70-76, wherein the subject has a gene mutation associated with heart disease, optionally wherein the gene mutation is a mutation in PLN. 78. The method of any one of claims 70-77, wherein the subject has low or undetectable levels of polypeptide expression compared to a healthy subject. 1. A method of treating or preventing a cardiac disease or disorder in a subject in need thereof, said method comprising the vector of claim 53, the rAAV virion of any of claims 54 to 61, or the composition of claim 62. A method comprising administering to the subject. 80. The method of claim 79, wherein the subject has a cardiac disease or disorder. 80. The method of claim 79, wherein the subject is at risk of developing a cardiac disease or disorder. 82. The method of any one of claims 79-81, wherein the heart disease or disorder is cardiomyopathy. The method of claim 82, wherein the cardiomyopathy is dilated cardiomyopathy. 82. The method of any one of claims 79-81, wherein the heart disease or disorder is myocardial infarction. 85. The method of claim 84, wherein the myocardial infarction is chronic myocardial infarction. 85. The method of claim 84, wherein the myocardial infarction is acute myocardial infarction. 87. The method of any one of claims 79-86, wherein the subject has a genetic risk allele for a heart disease or disorder. 88. The method of claim 87, wherein the genetic risk allele comprises a mutation in the PLN gene, optionally wherein the mutation in the PLN gene is a PLN promoter mutation, a PLN gene duplication, an R14del mutation. 89. The method of any one of claims 79-88, wherein the cardiac disease or disorder is reduced ejection fraction (HFrEF). 89. The method of any one of claims 79-88, wherein the cardiac disease or disorder is preserved ejection fraction (HFpEF). 91. The method of any one of claims 79-90, wherein the method induces expression of the polypeptide in the heart of the subject. 92. The method of any one of claims 79-91, wherein the method induces expression of the polypeptide in cardiomyocytes of the subject. 93. The method of any one of claims 79-92, wherein the method does not result in detectable expression of the polypeptide in the subject's heart, liver, and/or muscles of the subject other than the subject's cardiac fibroblasts. 94. The method of any one of claims 79-93, wherein the method improves one or more measures of cardiac function, optionally fractional shortening and/or left ventricular internal dimension (LVID). The method of claim 94, wherein the improvement in cardiac function is at week 2, week 4, week 6, week 8, week 10, week 12, week 14, week 16, week 18, week 20, week 22, and/or week 24 after administration. Observed at or after parking, method. 96. The method of any one of claims 79-95, wherein the administration is systemic administration. 97. The method of claim 96, wherein systemic administration is selected from intravenous or intraluminal injection. 98. The method of claim 96 or 97, wherein the rAAV virions are administered as a unit dose. 98. The method of claim 98, wherein the unit dose is less than or equal to about 3Х10 ¹⁴ vg/kg, less than or equal to about 2Х10 ¹⁴ vg/kg, less than or equal to about 1Х10 ¹⁴ vg/kg, less than or equal to about 9Х10 ¹³ vg/kg, or less than or equal to about 8Х10 ¹³ vg/kg, About 7Х10 ¹³ vg/kg or less, about 6Х10 ¹³ vg/kg or less, about 5Х10 ¹³ vg/kg or less, about 4Х10 ¹³ vg/kg or less, about 3Х10 ¹³ vg/kg or less, about 2Х10 ¹³ vg/kg or less, or about Method containing not more than 1Х10 ¹³ vg/kg. The method of any one of claims 79 to 99, wherein the subject is a mammal. The method of any one of claims 79-99, wherein the subject is a human.